Cancer targets and uses thereof

ABSTRACT

Methods and compositions are provided for assessing, treating, and preventing diseases, especially cancer, using cancer-associated targets (“CAT”). Methods and compositions are also provided for determining or predicting the effectiveness of a treatment for these diseases or for selecting a treatment, using CAT. Methods and compositions are further provided for modulating cell function using CAT. Also provided are compositions that modulate CAT (e.g., antagonists or agonists), such as antibodies, proteins, small molecule compounds, and nucleic acid agents (e.g., RNAi and antisense agents), as well as pharmaceutical compositions thereof. Further provided are methods of screening for agents that modulate CAT, and agents identified by these screening methods.

FIELD OF THE INVENTION

This invention relates to the field of disease assessment and therapy.The invention provides compositions and methods for assessing andtreating diseases, especially cancer.

BACKGROUND OF THE INVENTION Cancer

Cancer is one of the leading causes of death worldwide, and cancer isdifficult to diagnose and treat effectively. Accordingly, there is aneed in the art for new compositions and methods for assessing andtreating various cancers.

Cancer Stem Cells (CSCs)

Cancer stem cells (CSCs), which may also be referred to as tumor stemcells, are cancer cells that are operationally similar to normal stemcells and that typically have the ability to grow (or re-grow) a tumor.For example, like normal stem cells, CSCs typically possess theproperties of self-renewal ability, extensive proliferation potential,and the potential to differentiate into multiple phenotypicallydifferent cell types. CSCs can potentially arise from, for example,mutation of a normal adult stem cell or from mutations that impart stemcell-like properties to another type of cell. Therefore, CSCs may or maynot originate from their normal stem cell counterpart. However,regardless of whether or not the cell of origin of a CSC is in fact atrue stem cell, the term CSC is used in the art on the basis of thecancer cell having properties that are similar to normal stem cells.

For example, recent studies have demonstrated that in many types ofcancer, only a small subset of cancer cells within a tumor have theability to proliferate extensively, to initiate the growth of new tumors(including metastatic growth), and to differentiate into the variousdifferent cell types that make up a typical, complex heterogeneoustumor. These cells are the CSCs, and these properties are typical ofCSCs. Some CSCs have previously been isolated (e.g., from leukemia, aswell as brain, breast, and lung cancers) and, when transplanted into ananimal model (or even serially passaged into multiple animals), havebeen shown to be necessary and sufficient to initiate the growth of newtumors, whereas the vast majority of other cells within a tumor do nothave the capability to initiate the growth of new tumors.

However, current cancer therapies (including, for example, chemotherapyand radiation therapy) generally attempt to non-discriminately killproliferating cells, and potential therapeutic agents are commonlyselected based on their ability to reduce tumor size. Furthermore,clinical trials of anti-cancer agents are commonly designed with theobjective of demonstrating a reduction in tumor size. However, becausecancer stem cells typically make up only a small proportion of a tumor,reduction in tumor size may not be indicative of any reduction in cancerstem cell populations, and therefore reduction in tumor size may notaccurately reflect longer-term cancer prognosis. Because CSCs typicallyrepresent only a minor portion of a tumor and can be quiescent(non-proliferating), cancer therapies may primarily kill other cellsthat make up the majority of the tumor, thereby often leading to tumorshrinkage and possibly temporary cancer remission or other clinicalimprovement. However, because the CSCs have not been directly targetedand killed, CSCs may survive the cancer therapy and eventually initiatere-growth of the cancer. Thus, to permanently eradicate a cancer and toprevent metastasis, it would be desirable to specifically kill the CSCs.Similarly, to gain a more comprehensive diagnosis of a cancer, such asin predicting the reoccurrence of a cancer following treatment or thethreat of metastasis, it would be desirable to specifically assess theCSCs.

Therefore, there is a need in the art to identify cancer stem cellmarkers (e.g., proteins expressed by cancer stem cells, and the encodingnucleic acid molecules), as well as agents (e.g., antibodies) thattarget these cancer stem cell markers, so that more effective cancertherapies and diagnostics can be implemented that specifically targetthe small population of tumor cells within a tumor that are cancer stemcells and which can be most harmful to a patient with regards to tumorgrowth, metastasis and initiation of new tumors, and re-growth of tumorsfollowing treatment.

For a further review of CSCs, see the following (each of thesereferences is incorporated herein by reference): Fang et al., “Atumorigenic subpopulation with stem cell properties in melanomas”,Cancer Res. 2005 Oct. 15; 65(20):9328-37; Lee et al., “Tumor stem cellsderived from glioblastomas cultured in bFGF and EGF more closely mirrorthe phenotype and genotype of primary tumors than do serum-cultured celllines”, Cancer Cell. 2006 May; 9(5):391-403; Nishizuka, “Profilingcancer stem cells using protein array technology”, Eur J Cancer. 2006June; 42(9):1273-1282; Reya et al., “Stem cells, cancer, and cancer stemcells”, Nature 2001 Nov. 1; 414(6859):105-11; Huff et al., “The paradoxof response and survival in cancer therapeutics”, Blood 2006 Jan. 15;107(2):431-4; Feinberg et al., “The epigenetic progenitor origin ofhuman cancer”, Nat Rev Genet 2006 January; 7(1):21-33; Al-Hajj et al.,“Therapeutic implications of cancer stem cells”, Curr Opin Genet Dev.2004 February; 14(1):43-7; Soltysova et al., “Cancer stem cells”,Neoplasma 2005; 52(6):435-40; Wang et al., “Cancer stem cells: lessonsfrom leukemia”, Trends Cell Biol. 2005 September; 15(9):494-501; Jordan,“Cancer stem cell biology: from leukemia to solid tumors”, Curr OpinCell Biol. 2004 December; 16(6):708-12; Liu et al., “Adult stem cellsand cancer stem cells: tie in or tear apart?”, J Cancer Res Clin Oncol.2005 October; 131(10):631-8; Bjerkvig et al., “Opinion: the origin ofthe cancer stem cell: current controversies and new insights”, Nat RevCancer 2005 November; 5(11):899-904; Brabletz et al., “Opinion:migrating cancer stem cells—an integrated concept of malignant tumourprogression”, Nat Rev Cancer 2005 September; 5(9):744-9; Jones et al.,“Cancer stem cells: are we missing the target?”, J Natl Cancer Inst.2004 Apr. 21; 96(8):583-5; Al-Hajj et al., “Self-renewal and solid tumorstem cells”, Oncogene 2004 Sep. 20; 23(43):7274-82; Kopper et al.,“Tumor stem cells”, Pathol Oncol Res. 2004; 10(2):69-73; Cheng, “Cellcycle inhibitors in normal and tumor stem cells”, Oncogene 2004 Sep. 20;23(43):7256-66; and U.S. Pat. No. 6,984,522 (“Isolation and Use of SolidTumor Stem Cells”).

Description of the Files Contained on the CD-R Named CL001733CDR

The CD-R named CL001733CDR contains the following three text (ASCII)files:

1) File SEQLIST_(—)1733ORD.txt provides the Sequence Listing. TheSequence Listing provides exemplary protein sequences (SEQ ID NOS:1-722,1975, and 1977), transcript sequences (SEQ ID NOS:723-1281, 1976, and1978), and peptide sequences (SEQ ID NOS:1282-1974) as shown in Table 1.File SEQLIST_(—)1733ORD.txt is 5,905 KB in size and was created on Nov.20, 2008.

2) File TABLE1_(—)1733.txt provides Table 1, which is 1,018 KB in sizeand was created on Nov. 20, 2007.

3) File TABLE2_(—)1733.txt provides Table 2, which is 14 KB in size andwas created on Nov. 20, 2007.

The material contained on the CD-R named CL001733CDR is herebyincorporated by reference pursuant to 37 CFR 1.77(b)(4).

LENGTHY TABLES The patent contains a lengthy table section. A copy ofthe table is available in electronic form from the USPTO web site(http://seqdata.uspto.gov/?pageRequest=docDetail&DocID=US08168586B1). Anelectronic copy of the table will also be available from the USPTO uponrequest and payment of the fee set forth in 37 CFR 1.19(b)(3).

Description of Table 1

Table 1 (provided on the CD-R) discloses cancer-associated target(“CAT”) proteins, transcripts, and peptides (each protein, transcript,and peptide is represented by a SEQ ID NO in Table 1, and thecorresponding sequence is provided in the Sequence Listing; the range ofnumbers in parentheses following each peptide SEQ ID NO represents theamino acid residues of the location of the peptide within itscorresponding protein), the disease (e.g., cancer) cell lines ortissues, the expression ratio (“ratio”) of the peptide in the diseasesample compared to a control sample, and the type of cancer (or, if“adipose” is indicated, adipose disease such as diabetes or obesity) inwhich differential expression of the target was observed (“disease”).

The expression ratio is based on measuring the expression level of thepeptides, which are fragments of each full-length protein. Thus, theexpression level of the peptides is indicative of the expression levelof the corresponding protein of which the peptide is a fragment.Numerical representation of over-expression is indicated by 2.0 or more,whereas numerical representation of under-expression is indicated by 0.5or less. Over-expressed singleton indicates that a peptide peak wasdetected in a disease (e.g., cancer) sample but no peak was detected ina control sample. Under-expressed singleton indicates that a peptidepeak was detected in a control sample but no peak was detected in adisease sample.

The protein/gene/transcript information provided for each targetincludes any or all of the information selected from the following:

-   -   a protein SEQ ID NO    -   an internal identification number for the protein (hCP and/or        UID)    -   a public protein accession number (Genbank, e.g., RefSeq NP        number, Swiss-prot, or Derwent) for the protein    -   a protein name recognized in the art    -   an internal identification number for the gene (hCG and/or UID)    -   an art-recognized gene symbol    -   OMIM (“Online Mendelian Inheritance in Man” database; Johns        Hopkins University/NCBI) gene/protein name(s) and/or symbol(s)    -   a transcript SEQ ID NO    -   an internal identification number for the transcript (hCT and/or        UID)    -   a public transcript accession number (Genbank, e.g., RefSeq NM        number, or Derwent)

Description of Table 2

Table 2 discloses tumor staging information for tumor tissue sampleslisted in Table 1, as follows (all tumor staging designations in Table 2are conventional in the art):

Sample ID Number (corresponding to a tumor tissue sample listed in Table1), sample type (labeled “Sample”), cancer type (labeled “Type”), LymphNodes (the “N” in the TNM staging system, for Node, which designates thespread of a tumor to the lymph nodes), Distant Metastasis (the “M” inthe TNM staging system, for Metastasis, which designates the extent oftumor metastasis), Extent of Invasion (the “T” in the TNM stagingsystem, for Tumor, which designates the size and location of a primarytumor), and AJCC Stage (which is assigned based on a combination of theT, N, and M classifications).

Description of Table 3

Table 3 summarizes results of expression and functional validationanalyses for the cancer-associated targets (“CAT”) provided herein.

The column labeled “Target Symbol” provides the target symbolidentifying the target, which can be used to cross-reference targetsbetween Table 3 and Table 1.

The column labeled “IHC” provides results of immunohistochemistryanalysis of target protein expression in tumor tissue samples. For IHCstudies, the expression of each target was typically evaluated in 10tumor specimen for 10-14 cancer indications and 2 normal specimens ofthe same tissue type. A pathology score was given to each specimen by acertified pathologist. The pathology scores were based on: (i) stainingintensity that ranged from 0-4, 0 being the weakest and 4 being thestrongest staining, and (ii) the number of cells staining positive; fora specimen to be considered positively stained, >25% percent of cellsneeded to have a staining score of 1 or above. Once the scores areassigned for each specimen, the percent of tumors that have 2 or morepathology scores above the highest normal is calculated. Thesepercentages are listed in Table 3 (in the column labeled “IHC”).

The columns labeled “Apoptosis” and “Proliferation” provide results ofapoptosis and cell proliferation functional assays, respectively, incancer cell lines in which each target was knocked down by RNAi. In theproliferation or apoptosis columns, a “+” indicates that the target hasbeen functionally validated based on an RNAi screen. Experimentally, apool of four RNAi duplexes against a target was transfected in one ormore cell lines for each indication. If the transfection results in >35%decrease in proliferation or >2-fold increase in apoptosis in a cellline, the results were considered significant and the target wasassigned a “+” notation (which is indicated in Table 3).

Once a cell line has passed a screen, further validation steps wereundertaken. First, the screen positive cell line is transfected withmaximum concentration (100 nM) of each of four individual duplexes thatwere included in the pool in the above description. Typically, 1-2duplexes out of the pool of 4 will show similar effects on proliferationand/or apoptosis as the pool transfection. Effect of mRNA knockdown byindividual duplexes was also measured at this stage. Next, decreasingconcentrations of the selected duplex (100 nM down to 1 nM) weretransfected into the cell line to confirm specificity of the selectedduplex. If decreasing the duplex concentration resulted in decreasedeffect on proliferation and apoptosis of the cell line, the target wasconsidered to be functionally validated and was therefore assigned a“++” notation (which is indicated in Table 3).

For selected targets, if an antibody is commercially available or hasbeen custom generated, the effect of inhibition of the cell lineproliferation was measured in the presence of different concentrationsof the antibody and its isotype control. If the presence of an antibodyagainst a target showed inhibition of proliferation in one or more celllines, the target was considered functionally validated using anantibody and was therefore assigned a “+++” notation (which is indicatedin Table 3) (a target with a “+++” notation was not necessarily also“++” validated).

The column labeled “Matrix” refers to the experiments where thecomparisons were made between “case” and “control” to identify theover-expressed protein/peptides. Each matrix entry is identified by aprefix that defines the cell line and a suffix that defines thecomparison or culture condition used to generate the cell line. Asexamples,

1. H1299_Stemcell_Cys_only:

Spheroids (stem cells) generated from lung H1299 cell line were comparedto adherent cells obtained either from (i) primary culture or (ii)spheroid population; only cystine containing peptides were analyzed inthis experiment.

2. H1299_Stemcell_Glyco:

Spheroids (stem cells) generated from lung H1299 cell line were comparedto adherent cells obtained either from (i) primary culture or (ii)spheroid population; only peptides captured based on glycosylatedresidues were analyzed in this experiment.

3. CBT026_HES_Primary

Spheroids (stem cells) generated from colon tumor tissue CBT026T werecompared to the primary processed tumor tissue.

4. HT29_Stemcell:

Spheroids (stem cells) generated from colorectal cell line HT29 werecompared to adherent cells obtained from primary culture.

5. CBT026_T:

Spheroids (stem cells) generated from colon tumor tissue CBT026T werecompared to the adherent cells in culture obtained from the same tumortissue.

6. CBT026_N:

Spheroids (stem cells) generated from normal colon tissue CBT026N werecompared to the adherent cells in culture obtained from the same tissue.

7. RKO Sphere:

Spheroids (stem cells) generated from colorectal cell line RKO werecompared to adherent cells obtained from primary culture.

8. Melanoma Stemcell_EGT003D:

Spheroids (stem cells) generated from melanoma tumor tissue EGT003D werecompared to the adherent cells in culture obtained from the same tissue.

9. LS123_Stemcell:

Spheroids (stem cells) generated from colorectal cell line LS123 werecompared to adherent cells obtained from primary culture.

10. CBT026_(—)12Maps:

Spheroids (stem cells) generated from colon tumor tissue CBT026T andnormal tissue CBT026N were compared with adherent cells obtained fromculturing these tissues.

11. CBT026_five sets with N Primary:

Spheroids (stem cells) generated from colon tumor tissue CBT026T oradherent cells were compared to spheroids and adherent cells from normaltissue.

Further information about cell lines:

-   -   Lung metastatic non-small cell lung cancer cell line H1299        (catalog no. CRL-5803) was purchased through ATCC and cultured        according to the vendor recommendations.    -   Colorectal cancer cell lines HT-29 (Catalog no. HTB-39), LS123        (catalog number: CCL-255), and RKO (catalog no. CRL-2577) were        also obtained from ATCC and cultured according to the vendor        recommendations.    -   Spheroid cells (stem cells) were generated from colorectal tumor        tissue CBT026T (from 62 yr old male; grade: G2; stage: IIIC; TNM        Status: T4N2MX) and its adjacent normal colorectal tissue        CBT026N. The spheroid cell line generated from colorectal tumor        tissue CBT026T (which was used in comparisons that are        designated in the “Matrix” column of Table 3 as “CBT026_T” and        “CBT026_HES_Primary”) has been deposited with the American Type        Culture Collection (ATCC), 10801 University Boulevard, Manassas,        Va. 20110-2209, USA, under the terms of the Budapest Treaty on        the International Recognition of the Deposit of Microorganisms        for the Purpose of Patent Procedure on Jul. 21, 2006 and bears        the ATCC accession number PTA-7742.    -   Spheroid cells (stem cells) were generated from melanoma tumor        tissue EGT003D (identified in the “Matrix” column of Table 3 as        “Melanoma StemCell_EGT003D”).

The column labeled “Subcellular Location” indicates the cellularlocation of the target (either cell surface or secreted).

DESCRIPTION OF THE FIGURES

FIGS. 1-4. Results of experimental validation (TaqMan, RNAi, and, forANGPTL4, Flow Cytometry) for Angiopoietin-like 4 protein (ANGPTL4) (FIG.1), Tweety Homolog 3 (TTYH3) (FIG. 2), CEACAM6 (FIG. 3), and ProteinFLJ11273 (FIG. 4).

In the figures, with respect to RNAi validation results, “Apo.” refersto an apoptosis assay, and “Prol.” refers to a cell proliferation assay.“+”, “++”, and “−” indicate relative degree of RNAi-mediated effectobserved in the assay (either inducement of apoptosis or inhibition ofcell proliferation) in various cancer cell lines (as indicated), with“+” indicating that an RNAi-mediated effect was observed, “++”indicating that a strong RNAi-mediated effect was observed, and “−”indicating that an RNAi-mediated effect was not observed.

H1299 is a lung cancer cell line, CBT026T is a colon cancer cell line,and EGT003D is a melanoma cell line.

Cell lines labeled “DMEM” refer to differentiated (adherent) cell lines(DMEM indicates a type of culture medium suitable for the growth ofdifferentiated cells). Cell lines labeled “HES” and “HES-S” refer tocancer stem cell lines (HES and HES-S, which are used hereininterchangeably, indicate a type of culture media suitable for thegrowth of human embryonic stem, or “HES”, cells and cancer stem cells).Cell lines labeled “H/D” refer to differentiated cell populationsderived from cancer stem cells. Cell lines labeled “MBM4” refer toadherent melanoma cells generated in vitro from a primary tumor byculturing in serum-containing melanoma cell-derived MBM4 medium.

DETAILED DESCRIPTION OF EXEMPLARY EMBODIMENTS OF THE INVENTION

The invention will best be understood by reference to the followingdetailed description of the exemplary embodiments, taken in conjunctionwith the accompanying table(s) and/or figure(s). The discussion below isexemplary and is not to be taken as limiting the scope defined by theclaims.

The invention generally relates to molecules that have been identifiedusing proteomic analysis techniques such as MALDI-TOF/TOF LC/MS-basedprotein expression analysis to determine the expression levels ofproteins in disease tissues and/or disease cell lines (tissues and celllines may be collectively referred to as “samples”) and in normaltissues and/or normal cell lines, such that proteins that aredifferentially expressed (e.g., over- or under-expressed) in diseasesamples compared with normal samples are identified.

Exemplary embodiments of the invention provide the targets shown inTables 1 and 3 and methods of using these targets. Four of these targets(ANGPTL4, TTYH3, CEACAM6, and Protein FLJ11273) are also shown in thefigures and described in section 13 of the Examples section (which isentitled “Specific Examples of Results from Experimental Validation”).All of these targets (i.e., the targets provided in Table 1, Table 3,and/or the figures) are collectively referred to herein as “CAT”(“cancer-associated targets”). In particular, exemplary embodiments ofthe invention provide methods of using these targets for assessing(e.g., diagnosing, prognosing, or determining future susceptibility),treating, and preventing cancer. Each of these targets is associatedwith specific types of cancers in particular, as shown in Tables 1 and3, the figures, and/or described in section 13 of the Examples section.These targets also are expressed by cancer stem cells and thereforethese targets have utilities with respect to cancer stem cells, inaddition to their other cancer-related uses, such as for specificallytargeting cancer stems cells (e.g., for therapeutic or diagnosticpurposes) or for detecting, isolating, and/or labeling cancer stemcells.

Based on, for example, the finding that certain proteins, referred toherein as cancer-associated target (“CAT”) proteins, are differentiallyexpressed in cancer samples in comparison with normal samples, exemplaryembodiments of the invention provide methods and compositions forassessing, treating, and preventing diseases, especially cancer,particularly the cancers identified in Table 1 (as indicated for eachpeptide) and/or Table 3, using CAT. Furthermore, the compositions andmethods of the invention may be suitable for other types of cancer,particularly other epithelial cell-related cancers and solid tumors.

Cancer-associated target (“CAT”) proteins and fragments thereof (e.g.,CAT peptides), and CAT nucleic acid molecules and fragments thereofencoding CAT proteins and peptides, are collectively referred to as CATor “targets” (which may be interchangeably referred to as “markers” or“biomarkers”). Exemplary CAT proteins are provided as SEQ ID NOS:1-722,1975, and 1977, exemplary fragments of these CAT proteins are providedas the peptide sequences of SEQ ID NOS:1282-1974, and exemplary CATtranscript sequences (which encode the CAT proteins of SEQ ID NOS:1-722,1975, and 1977) are provided as SEQ ID NOS:723-1281, 1976, and 1978.These targets can be, for example, cell surface proteins, cytosolicproteins, or secreted proteins, as well as nucleic acid molecules thatencode these proteins.

The terms “protein” and “polypeptide” are used herein interchangeably.Exemplary CAT proteins/polypeptides are provided as SEQ ID NOS:1-722,1975, and 1977. A “peptide” typically refers to a fragment of aprotein/polypeptide. Thus, peptides are interchangeably referred to asfragments. Exemplary CAT peptides are provided as SEQ ID NOS:1282-1974.SEQ ID NOS:1282-1974 are fragments of SEQ ID NOS:1-722, 1975, and 1977(in Table 1, the range of numbers in parentheses following each peptideSEQ ID NO represents the amino acid residues of the location of thepeptide within its corresponding protein).

References herein to proteins, peptides, nucleic acid molecules, andantibodies typically are not limited to the full-size or full-lengthmolecule, but also can encompass fragments of these molecules (unless aparticular sequence or structure is explicitly stated).

Exemplary embodiments of the invention, which are discussed in greaterdetail below, provide antibodies, proteins, immunogenic peptides (e.g.,peptides which induce a T-cell response), or other biomolecules, as wellas small molecules, nucleic acid agents (e.g., RNAi and antisensenucleic acid agents), and other compositions that modulate the targets(e.g., agonists and antagonists), such as by binding to or otherwiseinteracting with or affecting the targets. These compositions can beused for assessing, treating, and preventing diseases, especiallycancer, particularly the cancers identified in Table 1 (as indicated foreach peptide) and/or Table 3, as well as other uses. Moreover, theinvention provides methods for assessing, treating, and preventingdiseases such as these based on CAT, such as by using thesecompositions. Further provided are methods of screening for agents thatmodulate CAT, such as by affecting the function, activity, and/orexpression level (“expression level” may be interchangeably referred toas “abundance level” or “level”) of CAT, and agents identified by thesescreening methods.

Exemplary embodiments of the invention also provide methods ofmodulating cell function. In particular, the invention provides methodsof modulating cell proliferation and/or apoptosis. For example, forcancer/tumor cells, the invention provides methods of inhibiting cellproliferation and/or stimulating apoptosis. Such methods can be appliedto the treatment of diseases, especially cancer.

Exemplary embodiments of the invention further provide methods ofdetermining or predicting effectiveness or response to a particulartreatment, and methods of selecting a treatment for an individual. Forexample, targets that are differentially expressed by cells that aremore or less responsive or resistant to a particular treatment, such asa cancer treatment, are useful for determining or predictingeffectiveness or response to the treatment or for selecting a treatmentfor an individual. Exemplary embodiments of the invention also providemethods of selecting individuals for a clinical trial of a therapeuticagent. For example, the targets can be used to identify individuals forinclusion in a clinical trial who are more likely to respond to aparticular treatment. Alternatively, the targets can be used to excludeindividuals from a clinical trial who are less likely to respond to aparticular treatment or who are more likely to experience toxic or otherundesirable side effects from a particular treatment.

Further exemplary embodiments of the invention are described in greaterdetail below.

Cancer-Associated Targets (CAT) as Cancer Stem Cell (CSC) Targets

The CAT proteins and encoding nucleic acid molecules provided in Table 1and/or Table 3 are expressed by cancer stem cells (“CSCs”) and thereforeare useful as CSC targets (as exemplified in the figures for certaintargets). Thus, the cancer-associated targets (“CAT”) provided hereinmay be interchangeably referred to herein as CSC targets. Because theCAT provided herein are expressed by cancer stem cells, the targetsprovided herein are particularly useful for cancer stem cell-relatedpurposes, such as for specifically targeting cancer stems cells (e.g.,for therapeutic or diagnostic purposes) or for detecting, isolating,and/or labeling cancer stem cells. For example, the targets providedherein are particularly useful as therapeutic targets for cancer. Atherapeutic agent can specifically target a CAT protein, such as in aheterogenous tumor cell population, in order to more effectively andefficiently (e.g., by enabling the use of a lower dose of a therapeuticagent, thereby minimizing side effects such as toxicity) treat a cancerby targeting a cancer stem cell.

CSCs may be cultured under conditions (e.g., using a serum-free culturemedium such as media designated for human embryonic stem cells) in whichthe CSCs proliferate as non-adherent 3-dimensional (“3D”) spheres,closely mimicking in vivo tumor growth. Hence, CSCs may beinterchangeably referred to herein as “cancer spheroid cells”,“spheroids”, or “tumorospheres”. These 3D cultures of CSCs, which can bestably maintained in vitro, are useful for therapeutic target discoveryand drug testing, among other uses.

Typical cancer cells or heterogeneous populations of cancer cells thatare not CSCs (i.e., non-stem cell-like cancer cells) may beinterchangeably referred to herein as “differentiated”, “adherent”, or“parental” cells (although “parental” cells typically more specificallyrefer to differentiated/adherent cells from which cancer stem cells arederived from), or as “counterparts” to cancer stem cells.

In certain exemplary embodiments of the invention, the CAT proteins orencoding nucleic acid molecules provided in Table 1 and/or Table 3 areused for selectively targeting CSCs (such as in a tumor or otherheterogenous population of cancer cells), such as for therapeutic,preventative, diagnostic, or prognostic purposes. For example, byselectively targeting a CSC target provided herein (e.g., with anantibody or small molecule compound that selectively binds to a proteinprovided herein, or a nucleic acid agent such as an RNAi or antisensemolecule that hybridizes to a nucleic acid molecule that encodes aprotein provided herein), proliferation or apoptosis of CSCs can bemodulated, such as to inhibit proliferation or stimulate apoptosis ofCSCs (such as for the purpose of killing CSCs).

Exemplary embodiments of the invention provide agents, such asantibodies, antibody-drug conjugates, detectably labeled antibodies,small molecule compounds, and nucleic acid agents, which selectivelytarget the CSC targets provided herein, as well as methods of usingthese agents. Exemplary methods of using these agents include, but arenot limited to, methods for modulating CSC proliferation or apoptosis(e.g., inhibiting CSC proliferation or stimulating CSC apoptosis), andmethods for the treatment, prevention, or diagnosis of cancer (such asmay be achieved by selectively targeting CSCs).

The CSC targets provided herein are also useful to screen drugcandidates for agents that selectively bind, interact with, or otherwisetarget CSCs. Accordingly, certain exemplary embodiments of the inventionprovide screening methods that utilize the CSC targets provided herein,as well as agents identified using these CSC-specific drug screeningmethods and methods of using these agents.

The CSC targets provided herein are also useful in methods of elicitinga CSC-specific immune response. For example, the CSC targets orfragments thereof can be used in the preparation of immunogens or cancervaccines that utilize CSC antigens, either for prophylactic ortherapeutic purposes. Thus, certain exemplary embodiments of theinvention provide immunogens or cancer vaccines that comprise one ormore CSC targets provided herein, or fragments thereof, and methods ofproducing and using such immunogens or cancer vaccines.

With respect to methods of using the CSC targets provided herein,examples of various embodiments of the invention include, but are notlimited to, methods for distinguishing functionally differentpopulations of cancer cells; methods for diagnosing an individual withcancer; methods for predicting an individual's likelihood of developingcancer in the future; methods for determining the severity of cancer orpredicting cancer progression (e.g., likelihood and extent ofmetastasis, or rapidity and extent of tumor growth); methods fordetermining the effects of therapeutic agents (e.g., antibodies, smallmolecules, proteins, nucleic acid compositions such as antisense or RNAinucleic acids, etc.) on tumors or the interaction of these agents withCSCs; methods for selecting a therapeutic strategy (e.g., based onpredicted response of the cancer to a particular therapeutic agent);methods for screening, identifying, and testing therapeutic agents thattarget cancer stem cells; etc.

Thus, the CSC targets are useful in the diagnosis, prognosis, treatment,or prevention of cancer, particularly by selectively targeting CSCs, orfor screening for therapeutic or diagnostic agents for cancer (e.g.,small molecule compounds or antibodies that selectively bind to the CSCtargets), particularly therapeutic or diagnostic agents that targetCSCs. Agents that target CSC targets, such as antibodies, antibody-drugconjugates, and small molecule compounds, can be used to inhibitproliferation or stimulate apoptosis of CSCs. Accordingly, agents suchas these that target CSC targets can be used to treat cancer.Furthermore, agents that target (e.g., selectively bind to) CSC targetscan be used for such purposes as, for example, diagnostic or prognosticpurposes, for selecting and/or prescribing a therapeutic agent to beadministered to an individual or selecting a treatment regimen for anindividual, for monitoring or predicting an individual's response to aparticular treatment, for monitoring cancer progression or remission,for determining or predicting cancer recurrence, or for determining aspecific stage, sub-type, or other classification or characteristic of acancer. Uses such as these can be achieved, for example, by using anagent (such as an antibody, for example, which may optionally be coupledto a detectable label) that selectively binds to a CSC target providedherein in order to, for example, determine the presence or abundance ofCSCs or CSC targets in a tumor or to determine the proportion of CSCsrelative to other (non-stem cell-like) cancer cells in a tumor. The CSCtargets provided herein are useful for, for example, isolating ordifferentiating CSCs (e.g., isolating CSCs from tumor tissue samples).

CSCs can typically be isolated or cultured using a serum-free culturemedium such as media designated for human embryonic stem cells. Forexample, CSC populations can be derived from their adherent parentalcells by culturing the adherent parental cells in a serum-free culturemedium such as that designated for human embryonic stem cells.Furthermore, CSC populations can typically be differentiated back toadherent cells upon exposure of the CSC populations to serum.

CSCs may demonstrate higher tumorigenic capacity or retaintumorigenicity (as assayed by an anchorage-independent assay) comparedwith their adherent cell counterparts. Furthermore, CSCs may demonstrateincreased resistance relative to their adherent counterparts tochemotherapeutic drugs (e.g., irinotecan, a first line treatment forcolon cancer), whereas their adherent counterparts undergo apoptosisand/or demonstrate growth inhibition when exposed to the same drug.

1. CAT Proteins

Exemplary embodiments of the invention provide isolated CAT proteinsthat consist of, consist essentially of, or comprise the amino acidsequences of SEQ ID NOS:1-722, 1975, and 1977 (which are encoded by thenucleotide sequences of SEQ ID NOS:723-1281, 1976, and 1978,respectively), as well as all obvious variants of these proteins andnucleic acid molecules that are within the art to make and use. Examplesof such obvious variants include, but are not limited to,naturally-occurring allelic variants, pre-processed or mature processedforms of a protein, non-naturally occurring recombinantly-derivedvariants, orthologs, and paralogs. Such variants can readily begenerated using art-known techniques in the fields of recombinantnucleic acid technology and protein biochemistry.

A protein is said to be “isolated” or “purified” when it issubstantially free of cellular material or free of chemical precursorsor other chemicals. CAT proteins can be purified to homogeneity or otherdegrees of purity. The level of purification can be based on theintended use. The primary consideration is that the preparation allowsfor the desired function of the protein, even if in the presence ofconsiderable amounts of other components.

In some uses, “substantially free of cellular material” includespreparations of a protein having less than about 30% (by dry weight)other proteins (i.e., contaminating protein), less than about 20% otherproteins, less than about 10% other proteins, or less than about 5%other proteins. When the protein is recombinantly produced, it can alsobe substantially free of culture medium, i.e., culture medium representsless than about 20% of the volume of the protein preparation.

The language “substantially free of chemical precursors or otherchemicals” includes preparations of a protein in which the protein isseparated from chemical precursors or other chemicals that are involvedin the protein's synthesis. In one embodiment, the language“substantially free of chemical precursors or other chemicals” includespreparations of a CAT protein having less than about 30% (by dry weight)chemical precursors or other chemicals, less than about 20% chemicalprecursors or other chemicals, less than about 10% chemical precursorsor other chemicals, or less than about 5% chemical precursors or otherchemicals.

Isolated CAT proteins can be purified from cells that naturally expressit, purified from cells that have been altered to express it(recombinant), or synthesized using known protein synthesis methods(e.g., Sambrook et al., Molecular Cloning: A Laboratory Manual. 3rd.ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.,(2001)). For example, a nucleic acid molecule encoding a CAT protein canbe cloned into an expression vector, the expression vector introducedinto a host cell, and the protein expressed in the host cell. Theprotein can then be isolated from the cells by an appropriatepurification scheme using standard protein purification techniques.

A CAT protein or fragment thereof can be attached to heterologoussequences to form chimeric or fusion proteins. Such chimeric and fusionproteins comprise a protein operatively linked to a heterologous proteinhaving an amino acid sequence not substantially homologous to theprotein. “Operatively linked” indicates that the protein and theheterologous protein are fused in-frame. The heterologous protein can befused to the N-terminus or C-terminus of the protein.

In some uses, the fusion protein does not affect the activity of theprotein per se. For example, the fusion protein can include, but is notlimited to, beta-galactosidase fusions, yeast two-hybrid GAL fusions,poly-His fusions, MYC-tagged, HI-tagged, and Ig fusions. Such fusionproteins, particularly poly-His fusions, can facilitate the purificationof recombinant CAT proteins. In certain host cells (e.g., mammalian hostcells), expression and/or secretion of a protein can be increased byusing a heterologous signal sequence.

A chimeric or fusion CAT protein can be produced by standard recombinantDNA techniques. For example, DNA fragments coding for different proteinsequences can be ligated together in-frame in accordance withconventional techniques. In another embodiment, a fusion gene can besynthesized by conventional techniques including automated DNAsynthesizers. Alternatively, PCR amplification of gene fragments can becarried out using anchor primers that give rise to complementaryoverhangs between two consecutive gene fragments that can subsequentlybe annealed and re-amplified to generate a chimeric gene sequence(Ausubel et al., Current Protocols in Molecular Biology, 1992-2006).Moreover, many expression vectors are commercially available thatalready encode a fusion moiety (e.g., a GST protein). A CAT-encodingnucleic acid can be cloned into such an expression vector such that thefusion moiety is linked in-frame to the CAT protein.

To determine the percent identity of two amino acid sequences or twonucleic acid sequences, the sequences can be aligned for optimalcomparison purposes (e.g., gaps can be introduced in one or both of afirst and a second amino acid or nucleic acid sequence for optimalalignment and non-homologous sequences can be disregarded for comparisonpurposes). In an exemplary embodiment, at least 30%, 40%, 50%, 60%, 70%,80%, or 90% or more of the length of a reference sequence can be alignedfor comparison purposes. The amino acid residues or nucleotides atcorresponding amino acid positions or nucleotide positions can then becompared. When a position in the first sequence is occupied by the sameamino acid residue or nucleotide as the corresponding position in thesecond sequence, then the molecules are identical at that position (asused herein, amino acid or nucleic acid “identity” is equivalent toamino acid or nucleic acid “homology”). The percent identity between thetwo sequences is a function of the number of identical positions sharedby the sequences, taking into account the number of gaps, and the lengthof each gap, that are introduced for optimal alignment of the twosequences.

The comparison of sequences and determination of percent identity andsimilarity between two sequences can be accomplished using amathematical algorithm. (Computational Molecular Biology, Lesk, A. M.,ed., Oxford University Press, New York, 1988; Biocomputing: Informaticsand Genome Projects, Smith, D. W., ed., Academic Press, New York, 1993;Computer Analysis of Sequence Data, Part 1, Griffin, A. M., and Griffin,H. G., eds., Humana Press, N.J., 1994; Sequence Analysis in MolecularBiology, von Heinje, G., Academic Press, 1987; and Sequence AnalysisPrimer, Gribskov, M. and Devereux, J., eds., Stockton Press, New York,1991). In an exemplary embodiment, the percent identity between twoamino acid sequences is determined using the Needleman and Wunsch (J.Mol. Biol. (48):444-453 (1970)) algorithm which has been incorporatedinto the GAP program in the GCG software package, using either a Blossom62 matrix or a PAM250 matrix, a gap weight of 16, 14, 12, 10, 8, 6, or4, and a length weight of 1, 2, 3, 4, 5, or 6. In another exemplaryembodiment, the percent identity between two nucleotide sequences can bedetermined using the GAP program in the GCG software package (Devereuxet al., Nucleic Acids Res. 12(1):387 (1984)) using a NWSgapdna.CMPmatrix and a gap weight of 40, 50, 60, 70, or 80, and a length weight of1, 2, 3, 4, 5, or 6. In another exemplary embodiment, the percentidentity between two amino acid or nucleotide sequences is determinedusing the algorithm of E. Myers and W. Miller (CABIOS, 4:11-17 (1989))which has been incorporated into the ALIGN program (version 2.0), usinga PAM120 weight residue table, a gap length penalty of 12, and a gappenalty of 4.

The sequences of the proteins and nucleic acid molecules of theinvention can further be used as a “query sequence” to perform a searchagainst sequence databases to, for example, identify other proteinfamily members or related sequences. Such searches can be performedusing the NBLAST and XBLAST programs (version 2.0) of Altschul et al.(J. Mol. Biol. 215:403-10 (1990)). BLAST nucleotide searches can beperformed with the NBLAST program, score=100, wordlength=12, to obtainnucleotide sequences homologous to the query nucleic acid molecule.BLAST protein searches can be performed with the XBLAST program,score=50, wordlength=3, to obtain amino acid sequences homologous to thequery proteins. To obtain gapped alignments for comparison purposes,Gapped BLAST can be utilized as described in Altschul et al. (NucleicAcids Res. 25(17):3389-3402 (1997)). When utilizing BLAST and gappedBLAST programs, the default parameters of the respective programs (e.g.,XBLAST and NBLAST) can be used.

As used herein, two proteins (or a region or domain of the proteins)have significant homology/identity (also referred to as substantialhomology/identity) when the amino acid sequences are typically at leastabout 70-80%, 80-90%, 90-95%, 96%, 97%, 98%, or 99% identical Asignificantly homologous amino acid sequence can be encoded by a nucleicacid molecule that hybridizes to a CAT protein-encoding nucleic acidmolecule under stringent conditions, as more fully described below.

Orthologs of a CAT protein typically have some degree of significantsequence homology to at least a portion of a CAT protein and are encodedby a gene from another organism. Preferred orthologs are isolated frommammals, preferably non-human primates, for the development of humantherapeutic targets and agents. Such orthologs can be encoded by anucleic acid molecule that hybridizes to a CAT protein-encoding nucleicacid molecule under moderate to stringent conditions, as more fullydescribed below, depending on the degree of relatedness of the twoorganisms yielding the proteins.

Non-naturally occurring variants of the CAT proteins can readily begenerated using recombinant techniques. Such variants include, but arenot limited to, deletions, additions, and substitutions in the aminoacid sequence of the CAT protein. For example, one class ofsubstitutions is conserved amino acid substitutions. Such substitutionsare those that substitute a given amino acid in a CAT protein by anotheramino acid of like characteristics. Typically seen as conservativesubstitutions are the replacements, one for another, among the aliphaticamino acids Ala, Val, Leu, and Ile; interchange of the hydroxyl residuesSer and Thr; exchange of the acidic residues Asp and Glu; substitutionbetween the amide residues Asn and Gln; exchange of the basic residuesLys and Arg; and replacements among the aromatic residues Phe and Tyr.Guidance concerning which amino acid changes are likely to bephenotypically silent are found in Bowie et al., Science 247:1306-1310(1990).

Variant CAT proteins can be fully functional or can lack function in oneor more activities, e.g., ability to bind substrate, ability tophosphorylate substrate, ability to mediate signaling, etc. Fullyfunctional variants typically contain only conservative variations orvariation in non-critical residues or in non-critical regions.

Non-functional variants typically contain one or more non-conservativeamino acid substitutions, deletions, insertions, inversions, ortruncations, or a substitution, insertion, inversion, or deletion in acritical residue or critical region.

Amino acids that are essential for function can be identified by methodsknown in the art, such as site-directed mutagenesis or alanine-scanningmutagenesis (Cunningham et al., Science 244:1081-1085 (1989)). Thelatter procedure introduces single alanine mutations at every residue inthe molecule. The resulting mutant molecules are then tested forbiological activity or in assays such as in vitro proliferativeactivity. Sites that are critical for binding partner/substrate bindingcan also be determined by structural analysis such as crystallization,nuclear magnetic resonance, or photoaffinity labeling (Smith et al., J.Mol. Biol. 224:899-904 (1992); de Vos et al., Science 255:306-312(1992)).

Exemplary embodiments of the invention provide fragments of a CAT, andpeptides that comprise and consist of such fragments. An exemplaryfragment typically comprises at least about 5, 6, 8, 10, 12, 14, 16, 18,20 or more contiguous amino acid residues of a CAT protein. Suchfragments can be chosen based on the ability to retain one or more ofthe biological activities of CAT or can be chosen for the ability toperform a function, e.g., bind a substrate or act as an immunogen.Particularly important fragments are biologically active fragments, suchas peptides that are, for example, about 8 or more amino acids inlength. Such fragments can include a domain or motif of a CAT, e.g., anactive site, a transmembrane domain, or a binding domain. Further,possible fragments include, but are not limited to, soluble peptidefragments and fragments containing immunogenic structures. Domains andfunctional sites can readily be identified, for example, by computerprograms well known and readily available to those of skill in the art(e.g., PROSITE analysis).

Proteins can contain amino acids other than the 20 amino acids commonlyreferred to as the 20 naturally-occurring amino acids. Further, manyamino acids, including the terminal amino acids, can be modified bynatural processes, such as processing and other post-translationalmodifications, or by chemical modification techniques well known in theart. Common modifications that occur naturally in proteins are wellknown to those of skill in the art.

Known modifications include, but are not limited to, acetylation,acylation, ADP-ribosylation, amidation, covalent attachment of flavin,covalent attachment of a heme moiety, covalent attachment of anucleotide or nucleotide derivative, covalent attachment of a lipid orlipid derivative, covalent attachment of phosphotidylinositol,cross-linking, cyclization, disulfide bond formation, demethylation,formation of covalent crosslinks, formation of cystine, formation ofpyroglutamate, formylation, gamma carboxylation, glycosylation, GPIanchor formation, hydroxylation, iodination, methylation,myristoylation, oxidation, proteolytic processing, phosphorylation,prenylation, racemization, selenoylation, sulfation, tRNA-mediatedaddition of amino acids to proteins such as arginylation, andubiquitination.

Such modifications are well known to those of skill in the art and havebeen described in the scientific literature. Several particularly commonmodifications, glycosylation, lipid attachment, sulfation,gamma-carboxylation of glutamic acid residues, hydroxylation andADP-ribosylation, for instance, are described in most basic texts, suchas Proteins-Structure and Molecular Properties, 2nd Ed., T. E.Creighton, W.H. Freeman and Company, New York (1993). Many detailedreviews are available on this subject, such as by Wold(Posttranslational Covalent Modification of Proteins, B. C. Johnson,Ed., Academic Press, New York 1-12 (1983)); Seifter et al. (Meth.Enzymol. 182: 626-646 (1990)); and Rattan et al. (Ann. N.Y. Acad. Sci.663:48-62 (1992)).

Accordingly, exemplary CAT proteins and fragments thereof of theinvention can also encompasses derivatives or analogs in which, forexample, a substituted amino acid residue is not one encoded by thegenetic code, in which a substituent group is included, in which amature CAT is fused with another composition, such as a composition toincrease the half-life of a CAT (e.g., polyethylene glycol or albumin),or in which additional amino acids are fused to a mature CAT, such as aleader or secretory sequence or a sequence for purification of a matureCAT or a pro-protein sequence.

2. Antibodies to CAT Proteins

Exemplary embodiments of the invention provide antibodies to CATproteins, including, for example, monoclonal and polyclonal antibodies;chimeric, humanized, and fully human antibodies; and antigen-bindingfragments and variants thereof, as well as other embodiments.

Antibodies that selectively bind to a CAT protein can be made usingstandard procedures known to those of ordinary skills in the art. Theterm “antibody” is used in the broadest sense, and specifically covers,for example, monoclonal antibodies, polyclonal antibodies, multispecificantibodies (e.g., bispecific antibodies), chimeric antibodies, humanizedantibodies, fully human antibodies, and antibody fragments (e.g., Fab,F(ab′)₂, Fv and Fv-containing binding proteins), so long as they exhibitthe desired biological activity. Antibodies (Ab's) and immunoglobulins(Ig's) are glycoproteins typically having the same structuralcharacteristics. While antibodies exhibit binding specificity to aspecific antigen, immunoglobulins include both antibodies and otherantibody-like molecules that lack antigen specificity. Antibodies can beof the IgG, IgE, IgM, IgD, and IgA class or subclass thereof (e.g.,IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2). Antibodies may beinterchangeably referred to as “antigen-binding molecules”.

The term “monoclonal antibody”, as used herein, refers to an antibodyobtained from a population of substantially homogeneous antibodies,i.e., the individual antibodies comprising the population aresubstantially identical except for possible naturally occurringmutations that may be present in minor amounts. Monoclonal antibodiesare highly specific and are typically directed against a singleantigenic site. Furthermore, in contrast to polyclonal antibodypreparations, which typically include different antibodies directedagainst different determinants (epitopes), each monoclonal antibody istypically directed against a single determinant on an antigen. Inaddition to their specificity, monoclonal antibodies are advantageous inthat substantially homogenous antibodies can be produced by a hybridomaculture which is uncontaminated by other immunoglobulins or antibodies.The modifier “monoclonal” antibody indicates the character of theantibody as being obtained from a substantially homogeneous populationof antibodies, and is not to be construed as requiring production of theantibody by any particular method. For example, monoclonal antibodiescan be made by hybridoma methods such as described by Kohler andMilstein, Nature 256: 495-497 (1975), by recombinant methods (e.g., asdescribed in U.S. Pat. No. 4,816,567), or can be isolated from phageantibody libraries such as by using the techniques described in Clacksonet al., Nature 352: 624-628 (1991) or Marks et al., J. Mol. Biol. 222:581-597 (1991).

“Humanized” forms of non-human (e.g., murine or rabbit) antibodies arechimeric immunoglobulins, immunoglobulin chains or fragments thereof(such as Fv, Fab, Fab′, F(ab′)₂ or other antigen-binding subsequences ofantibodies) which contain minimal sequence derived from non-humanimmunoglobulin. Typically, humanized antibodies are humanimmunoglobulins (a recipient antibody) in which residues from acomplementarity determining regions (“CDR”) of the recipient arereplaced by residues from a CDR of a non-human species (a donorantibody) such as mouse, rat, or rabbit having the desired specificity,affinity, and capacity. In some instances, Fv framework residues of thehuman immunoglobulin are replaced by corresponding non-human residues.Furthermore, a humanized antibody may comprise residues which are foundneither in the recipient antibody nor in the imported CDR or frameworkregion (FR) sequences. These modifications can be made to further refineand optimize antibody performance. In general, a humanized antibody cancomprise substantially all of at least one, and typically two, variabledomains, in which all or substantially all of the CDRs correspond tothose of a non-human immunoglobulin and all or substantially all of theFRs are those of a human immunoglobulin consensus sequence. A humanizedantibody can also comprise at least a portion of an immunoglobulinconstant region (Fc), typically that of a human immunoglobulin. Forfurther details concerning humanized antibodies, see: Jones et al.,Nature 321:522-525 (1986); Reichmann et al., Nature 332:323-327 (1988);Presta, Curr. Op. Struct. Biol. 2:593-596 (1992); Queen et al., U.S.Pat. Nos. 5,530,101; 5,585,089; 5,693,762; and 6,180,370; and Winter,U.S. Pat. No. 5,225,539.

Antibodies, as used herein, include antibody fragments, particularlyantigen-binding fragments, as well as other modified antibody structuresand antigen-binding scaffolds (such as modified antibody structures thatare smaller or have less than all domains or chains compared with atypical naturally occurring, full-size human antibody). Examples ofantibody fragments and other modified antibody structures andantigen-binding scaffolds are known in the art by such terms asminibodies (e.g., U.S. Pat. No. 5,837,821), Nanobodies (llama heavychain antibodies; Ablynx, Ghent, Belgium), Adnectins (fibronectindomains; Adnexus Therapeutics, Waltham, Mass.), Affibodies(protein-binding domain of Staphylococcus aureus protein A; Affibody,Stockholm, Sweden), peptide aptamers (synthetic peptides; Aptanomics,Lyon, France), Avimers (A-domains derived from cell surface receptors;Avidia, Mountain View, Calif. (acquired by Amgen)), Transbodies(transferrin; BioRexis Pharmaceuticals, King of Prussia, Pa. (acquiredby Pfizer)), trimerized tetranectin domains (Borean Pharma, Aarhus,Denmark), Domain antibodies (heavy or light chain antibodies; Domantis,Cambridge, UK (acquired by GlaxoSmithKline)), Evibodies (derived fromV-like domains of T-cell receptors CTLA-4, CD28 and inducible T-cellcostimulator; EvoGenix Therapeutics, Sydney, Australia), scFV fragments(stable single chain antibody fragments; ESBATech, Zurich, Switzerland),Unibodies (monovalent IgG4 mAbs fragments; Genmab, Copenhagen, Denmark),BiTEs (bispecific, T-cell activating single-chain antibody fragments;Micromet, Munich, Germany), DARPins (designed ankyrin repeat proteins;Molecular Partners, Zurich, Switzerland), Anticalins (derived fromlipocalins; Pieris, Freising-Weihenstephan, Germany), Affilins (derivedfrom human lens protein gamma crystalline; Scil Proteins, Halle,Germany), and SMIPs (small modular immunopharmaceuticals; TrubionPharmaceuticals, Seattle, Wash.) (Sheridan, Nature Biotechnology, 2007April; 25(4):365-6).

An “isolated” or “purified” antibody is one that has been identified andseparated and/or recovered from a component of the environment in whichit is produced. Contaminant components of its production environment arematerials that would interfere with diagnostic or therapeutic uses forthe antibody, and may include enzymes, hormones, and other proteinaceousor nonproteinaceous solutes. In exemplary embodiments, the antibody canbe purified as measurable by any of at least three different methods: 1)to greater than 95% by weight of antibody as determined by the Lowrymethod, preferably more than 99% by weight; 2) to a degree sufficient toobtain at least 15 residues of N-terminal or internal amino acidsequence by use of a spinning cup sequenator; or 3) to homogeneity bySDS-PAGE under reducing or non-reducing conditions using Coomasie blueor silver stain. Isolated antibody can include an antibody in situwithin recombinant cells since at least one component of the antibody'snatural environment will not be present. Ordinarily, however, anisolated antibody can be prepared by at least one purification step.

An “antigenic region”, “antigenic determinant”, or “epitope” includesany protein determinant capable of specific binding to an antibody. Thisis the site on an antigen to which each distinct antibody moleculebinds. Epitopic determinants can be active surface groupings ofmolecules such as amino acids or sugar side chains and may have specificthree-dimensional structural characteristics or charge characteristics.

“Antibody specificity” refers to an antibody that has a stronger bindingaffinity for an antigen from a first subject species than it has for ahomologue of that antigen from a second subject species. Typically, anantibody “binds specifically” to a human antigen (e.g., has a bindingaffinity (Kd) value of no more than about 1×10⁻⁷ M, preferably no morethan about 1×10⁻⁸M, and most preferably no more than about 1×10⁻⁹ M) buthas a binding affinity for a homologue of the antigen from a secondsubject species which is at least about 50-fold, or at least about500-fold, or at least about 1000-fold, weaker than its binding affinityfor the human antigen. The antibodies can be of any of the various typesof antibodies as described herein, such as humanized or fully humanantibodies.

An antibody “selectively” or “specifically” binds a target protein whenthe antibody binds the target protein and does not significantly bind tounrelated proteins. An antibody can still be considered to selectivelyor specifically bind a target protein even if it also binds to otherproteins that are not substantially homologous with the target proteinas long as such proteins share homology with a fragment or domain of thetarget protein. In this case, it would be understood that antibodybinding to the target protein is still selective despite some degree ofcross-reactivity.

Exemplary embodiments of the invention provide an “antibody variant”,which refers to an amino acid sequence variant of an antibody whereinone or more of the amino acid residues have been modified. Such variantsnecessarily have less than 100% sequence identity with the amino acidsequence of the antibody, and have at least 75%, 80%, 85%, 90%, 95%,96%, 97%, 98%, or 99% amino acid sequence identity with the amino acidsequence of either the heavy or light chain variable domain of theantibody.

The term “antibody fragment” refers to a portion of a full-lengthantibody, including the antigen binding or variable region or theantigen-binding portion thereof. Examples of antibody fragments includeFab, Fab′, F(ab′)₂ and Fv fragments. Papain digestion of antibodiestypically produces two identical antigen binding fragments, called theFab fragment, each with a single antigen binding site, and a residual“Fc” fragment. Pepsin treatment typically yields an F(ab′)₂ fragmentthat has two antigen binding fragments which are capable of crosslinkingantigen, and a residual other fragment (which is termed pFc′). Examplesof additional antigen-binding fragments can include diabodies,triabodies, tetrabodies, single-chain Fv, single-chain Fv-Fc, SMIPs, andmultispecific antibodies formed from antibody fragments. A “functionalfragment”, with respect to antibodies, typically refers to an Fv, F(ab),F(ab′)₂ or other antigen-binding fragments comprising one or more CDRsthat has substantially the same antigen-binding specificity as anantibody.

An “Fv” fragment is an example of an antibody fragment that contains acomplete antigen recognition and binding site. This region typicallyconsists of a dimer of one heavy and one light chain variable domain ina tight, non-covalent association (V_(H)-V_(L) dimer). It is in thisconfiguration that the three CDRs of each variable domain interact todefine an antigen-binding site on the surface of the V_(H)-V_(L) dimer.Collectively, the six CDRs confer antigen-binding specificity to theantibody. However, even a single variable domain (or half of an Fvcomprising only three CDRs specific for an antigen) has the ability torecognize and bind antigen.

An “Fab” fragment (also designated as “F(ab)”) also contains theconstant domain of the light chain and the first constant domain (CH1)of the heavy chain. Fab′ fragments differ from Fab fragments by theaddition of a few residues at the carboxyl terminus of the heavy chainCH1 domain, including one or more cysteines from the antibody hingeregion. Fab′-SH is the designation for Fab′ in which the cysteineresidue(s) of the constant domains have a free thiol group. F(ab′)fragments are produced by cleavage of the disulfide bond at the hingecysteines of the F(ab′)₂ pepsin digestion product. Additional chemicalcouplings of antibody fragments are known to those of ordinary skill inthe art.

A “single-chain Fv” or “scFv” antibody fragment contains V_(H) and V_(L)domains, wherein these domains are present in a single polypeptidechain. Typically, the Fv polypeptide further comprises a polypeptidelinker between the V_(H) and V_(L) domains that enables the scFv to formthe desired structure for antigen binding. For a review of scFv, seePlückthun in The Pharmacology of Monoclonal Antibodies, vol. 113,Rosenburg and Moore eds., Springer-Verlag, New York, pp. 269-315 (1994).A single chain Fv-Fc is an scFv linked to a Fc region.

A “diabody” is a small antibody fragment with two antigen-binding sites,which fragments comprise a variable heavy domain (V_(H)) connected to avariable light domain (V_(L)) in the same polypeptide chain. By using alinker that is too short to allow pairing between the two domains on thesame chain, the domains are forced to pair with the complementarydomains of another chain and create two antigen-binding sites. Diabodiesare described more fully in, for example, EP 0 404 097; WO 93/11161; andHollinger et al., Proc. Natl. Acad. Sci. USA 90:6444-6448 (1993).Triabodies, tetrabodies and other antigen-binding antibody fragmentshave been described by Hollinger and Hudson, 2005, Nature Biotechnology23:1126.

A “small modular immunopharmaceutical” (or “SMIP”) is a single-chainpolypeptide including a binding domain (e.g., an scFv or an antigenbinding portion of an antibody), a hinge region, and an effector domain(e.g., an antibody Fc region or a portion thereof). SMIPs are describedin published U.S. Patent Application No. 20050238646.

Many methods are known for generating and/or identifying antibodies to agiven target protein. Several such methods are described by Kohler etal., 1975, Nature 256: 495-497; Lane, 1985, J. Immunol. Meth.81:223-228; Harlow et al., 1988, Antibodies: A Laboratory Manual. ColdSpring Harbor Laboratory Press; Harlow et al., 1998, Using Antibodies,Cold Spring Harbor Press; Zhong et al., 1997, J. Indust. Microbiol.Biotech. 19(1):71-76; and Berry et al., 2003, Hybridoma and Hybridomics22(1): 23-31.

Polyclonal antibodies can be prepared by any known method ormodifications of these methods, including obtaining antibodies frompatients. In certain exemplary methods for generating antibodies such aspolyclonal antibodies, an isolated protein can be used as an immunogenwhich is administered to a mammalian organism, such as a rat, rabbit, ormouse. For example, a complex of an immunogen such as a CAT protein (orfragment thereof) and a carrier protein can be prepared and an animalimmunized by the complex. Serum or plasma containing antibodies againstthe protein can be recovered from the immunized animal and theantibodies separated and purified (in the same manner as for monoclonalantibodies, for example). The gamma globulin fraction or the IgGantibodies can be obtained, for example, by use of saturated ammoniumsulfate or DEAE SEPHADEX, or other techniques known to those skilled inthe art. The antibody titer in the antiserum can be measured in the samemanner as in the supernatant of a hybridoma culture.

A full-length CAT protein, an antigenic peptide fragment, or a fusionprotein thereof, can be used as an immunogen. A protein used as animmunogen is not limited to any particular type of immunogen. In oneaspect, antibodies can be prepared from regions or discrete fragments(e.g., functional domains, extracellular domains, or portions thereof)of a CAT protein. Antibodies can be prepared from any region of aprotein as described herein. In particular, the proteins can be selectedfrom the group consisting of SEQ ID NOS:1-722, 1975, and 1977 andfragments thereof. An antigenic fragment can typically comprise at least8, 10, 12, 14, 16, or more contiguous amino acid residues, for example.Such fragments can be selected based on a physical property, such asfragments that correspond to regions located on the surface of a protein(e.g., hydrophilic regions) or can be selected based on sequenceuniqueness.

Antibodies can also be produced by inducing production in a lymphocytepopulation or by screening antibody libraries or panels of highlyspecific binding reagents, such as disclosed in Orlandi et al. (Proc.Natl. Acad. Sci. 86:3833-3837 (1989)) or Winter et al. (Nature349:293-299 (1991)). A protein can be used in screening assays ofphagemid or B-lymphocyte immunoglobulin libraries to identify antibodieshaving a desired specificity. Numerous protocols for competitive bindingor immunoassays using either polyclonal or monoclonal antibodies withestablished specificities are well known in the art (e.g., Smith, Curr.Opin. Biotechnol. 2: 668-673 (1991)).

Antibodies can also be generated using various phage display methodsknown in the art. In representative phage display methods, functionalantibody domains are displayed on the surface of phage particles whichcarry nucleic acid molecules that encode the antibody domains. Inparticular, such phage can be utilized to display antigen-bindingdomains expressed from a repertoire or combinatorial antibody library(e.g., human or murine). Phage expressing an antigen binding domain thatbinds an antigen of interest can be selected or identified with theantigen, e.g., using labeled antigen or antigen bound or captured to asolid surface or bead. Phage used in methods such as these can typicallybe filamentous phage including fd and M13 binding domains expressed fromphage with Fab, Fv, or disulfide stabilized Fv antibody domainsrecombinantly fused to either the phage gene III or gene VIII protein.Examples of phage display methods that can be used to make antibodiesinclude methods described in Brinkman et al., J. Immunol. Methods182:41-50 (1995); Ames et al., J. Immunol. Methods 184:177-186 (1995);Kettleborough et al., Eur. J. Immunol. 24:952-958 (1994); Persic et al.,Gene 187:9-18 (1997); Burton et al., Advances in Immunology 57:191-280(1994); PCT application No. PCT/GB91/01134; PCT publications WO90/02809; WO 91/10737; WO 92/01047; WO 92/18619; WO 93/11236; WO95/15982; WO 95/20401; and U.S. Pat. Nos. 5,698,426; 5,223,409;5,403,484; 5,580,717; 5,427,908; 5,750,753; 5,821,047; 5,571,698;5,427,908; 5,516,637; 5,780,225; 5,658,727; 5,733,743 and 5,969,108;each of which is incorporated herein by reference in its entirety.

Antibodies, antigen binding fragments, and/or antibody variants can beproduced by recombinant and genetic engineering methods well known inthe art. For example, methods of expressing heavy and light chain genesin E. coli are described in PCT publication numbers WO901443, WO901443,and WO9014424, and in Huse et al., 1989 Science 246:1275-1281. Whenusing recombinant techniques, such as to produce an antibody variant,the antibody variant can be produced intracellularly, in the periplasmicspace, or directly secreted into the medium. If an antibody variant isproduced intracellularly, as a first step, the particulate debris,either host cells or lysed fragments, can be removed, for example, bycentrifugation or ultrafiltration. Carter et al. (Bio/Technology 10:163-167 (1992)) describe a procedure for isolating antibodies that aresecreted to the periplasmic space of E. coli. Briefly, cell paste can bethawed in the presence of sodium acetate (pH 3.5), EDTA, andphenylmethylsulfonylfluoride (PMSF) over about 30 minutes. Cell debriscan be removed by centrifugation. Where an antibody variant is secretedinto the medium, supernatants from such expression systems can first beconcentrated using a commercially available protein concentration filter(e.g., an Amicon or Millipore PELLICON ultrafiltration unit). A proteaseinhibitor such as PMSF can be included in any of the foregoing steps toinhibit proteolysis, and antibiotics can be included to prevent thegrowth of contaminating microorganisms.

An antibody composition prepared from cells can be purified using, forexample, affinity chromatography, hydroxylapatite chromatography, gelelectrophoresis, and/or dialysis. The suitability of protein A as anaffinity ligand typically depends on the species and isotype of theimmunoglobulin Fc domain of an antibody. Protein A can be used to purifyantibodies that are based on human delta1, delta2, or delta4 heavychains (Lindmark et al., J. Immunol Meth. 62: 1-13 (1983)). Protein Gcan be used for all mouse isotypes and for human delta3 (Guss et al.,EMBO J. 5: 1567-1575 (1986)). The matrix to which the affinity ligand isattached can be, for example, agarose or mechanically stable matricessuch as controlled pore glass or poly(styrenedivinyl)benzene. Where theantibody comprises a CH3 domain, the BAKERBOND ABX™ resin (J. T. Baker,Phillipsburg, N.J.) can be used for purification. Other exemplarytechniques for antibody purification include, but are not limited to,fractionation on an ion-exchange column, ethanol precipitation, reversephase HPLC, chromatography on silica, chromatography on heparinhepharos, chromatography on an anion or cation exchange resin (such as apolyaspartic acid column), chromatofocusing, SDS-PAGE, and ammoniumsulfate precipitation.

Following any preliminary purification step(s), contaminants in amixture containing an antibody of interest can be removed by low pHhydrophobic interaction chromatography using an elution buffer at a pHbetween about 2.5-4.5, preferably performed at low salt concentrations(e.g., from about 0-0.25M salt).

Full-length antibodies, as well as antibody fragments, can also beexpressed and isolated from bacteria such as E. coli, such as describedin Mazor et al., “Isolation of engineered, full-length antibodies fromlibraries expressed in Escherichia coli”, Nat Biotechnol. 2007 May;25(5):563-5 and Sidhu, “Full-length antibodies on display”, NatBiotechnol. 2007 May; 25(5):537-8.

Further details regarding antibodies are set forth in the following U.S.Pat. Nos. 6,248,516 (Winter et al.); 6,291,158 (Winter et al.);5,885,793 (Griffiths et al.); 5,969,108 (McCafferty et al.); 5,939,598(Kucherlapati et al.); 4,816,397 (Boss et al.); 4,816,567 (Cabilly etal.); 6,331,415 (Cabilly et al.); 5,770,429 (Lonberg et al.); 5,639,947(Hiatt et al.); and 5,260,203 (Ladner et al.), each of which isincorporated herein by reference, and in the following published U.S.patent applications: US20040132101 (Lazar et al.), US20050064514(Stavenhagen et al.), US20040261148 (Dickey et al.), and US20050014934(Hinton et al.), each of which is incorporated herein by reference.Antibody engineering is further described in Jain et al., “Engineeringantibodies for clinical applications”, Trends Biotechnol. 2007 July;25(7):307-16.

3. Antibody-Drug Conjugates to CAT Proteins

An antibody against CAT can be coupled (e.g., covalently bonded) to asuitable therapeutic agent (as further discussed herein) either directlyor indirectly (e.g., via a linker group). A direct reaction between anantibody and a therapeutic agent is possible when each possesses asubstituent capable of reacting with the other. For example, anucleophilic group, such as an amino or sulfhydryl group, on onemolecule may be capable of reacting with a carbonyl-containing group,such as an anhydride or an acid halide, or with an alkyl groupcontaining a good leaving group (e.g., a halide) on the other molecule.

Alternatively, it may be desirable to couple a therapeutic agent and anantibody via a linker group. A linker group can function as a spacer todistance an antibody from an agent in order to avoid interference withbinding capabilities. A linker group can also serve to increase thechemical reactivity of a substituent on an agent or an antibody, andthus increase the coupling efficiency. An increase in chemicalreactivity may also facilitate the use of agents, or functional groupson agents, which otherwise would not be possible.

A variety of bifunctional or polyfunctional reagents, both homo- andhetero-functional (such as those described in the catalog of the PierceChemical Co., Rockford, Ill.), can be employed as the linker group.Coupling can be effected, for example, through amino groups, carboxylgroups, sulfhydryl groups, or oxidized carbohydrate residues (e.g., U.S.Pat. No. 4,671,958).

Where a therapeutic agent is more potent when free from the antibodyportion of an immunoconjugate, it may be desirable to use a linker groupthat is cleavable during or upon internalization into a cell. A numberof different cleavable linker groups have been described. Mechanisms forthe intracellular release of an agent from these linker groups includecleavage by reduction of a disulfide bond (e.g., U.S. Pat. No.4,489,710), by irradiation of a photolabile bond (e.g., U.S. Pat. No.4,625,014), by hydrolysis of derivatized amino acid side chains (e.g.,U.S. Pat. No. 4,638,045), by serum complement-mediated hydrolysis (e.g.,U.S. Pat. No. 4,671,958), by protease cleavable linker (e.g., U.S. Pat.No. 6,214,345), and by acid-catalyzed hydrolysis (e.g., U.S. Pat. No.4,569,789).

It may be desirable to couple more than one agent to an antibody.Multiple molecules of an agent can be coupled to one antibody molecule,and more than one type of agent can be coupled to the same antibody. Forexample, about 1, 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, or 22 (or anyother number in-between) molecules of therapeutic agents can be coupledto an antibody. The average number or quantitative distribution oftherapeutic agent molecules per antibody molecule in a preparation ofconjugation reactions can be determined by conventional means such asmass spectroscopy, ELISA, or HPLC. Separation, purification, andcharacterization of homogeneous antibody-drug conjugates having acertain number of therapeutic agents conjugated thereto can be achievedby means such as reverse phase HPLC or electrophoresis (see, e.g.,Hamblett et al., Clinical Cancer Res. 10:7063-70 (2004).

Examples of suitable therapeutic agents that can be conjugated to anantibody include, but are not limited to, chemotherapeutic agents (e.g.,cytotoxic or cytostatic agents or immunomodulatory agents),radiotherapeutic agents, therapeutic antibodies, small molecule drugs,peptide drugs, immunomodulatory agents, differentiation inducers, andtoxins.

Examples of useful classes of cytotoxic or immunomodulatory agentsinclude, but are not limited to, antitubulin agents, auristatins, DNAminor groove binders, DNA replication inhibitors, alkylating agents(e.g., platinum complexes such as cis-platin, mono(platinum),bis(platinum) and tri-nuclear platinum complexes and carboplatin),anthracyclines, antibiotics, antifolates, antimetabolites, chemotherapysensitizers, duocarmycins, etoposides, fluorinated pyrimidines,ionophores, lexitropsins, nitrosoureas, platinols, pre-formingcompounds, purine antimetabolites, puromycins, radiation sensitizers,steroids, taxanes, topoisomerase inhibitors, vinca alkaloids, and thelike.

Examples of individual cytotoxic or immunomodulatory agents include, butare not limited to, androgen, anthramycin (AMC), asparaginase,5-azacytidine, azathioprine, bleomycin, busulfan, buthioninesulfoximine, calicheamicin or calicheamicin derivatives, camptothecin orcamptothecins derivatives, carboplatin, carmustine (BSNU), CC-1065,chlorambucil, cisplatin, colchicine, cyclophosphamide, cytidinearabinoside (cytarabine), cytochalasin B, dacarbazine, dactinomycin(formerly actinomycin), daunorubicin, decarbazine, docetaxel,doxorubicin, etoposide, estrogen, 5-fluordeoxyuridine, 5-fluorouracil,gemcitabine, gramicidin D, hydroxyurea, idarubicin, ifosfamide,irinotecan, lomustine (CCNU), maytansine, mechlorethamine, melphalan,6-mercaptopurine, methotrexate, mithramycin, mitomycin C, mitoxantrone,nitroimidazole, paclitaxel, palytoxin, plicamycin, procarbizine,rhizoxin, streptozotocin, tenoposide, 6-thioguanine, thioTEPA,topotecan, vinblastine, vincristine, vinorelbine, VP-16, and VM-26.

Examples of other suitable cytotoxic agents include, but are not limitedto, DNA minor groove binders (e.g., enediynes and lexitropsins, a CBIcompound; see also U.S. Pat. No. 6,130,237), duocarmycins, taxanes(e.g., paclitaxel and docetaxel), puromycins, vinca alkaloids, CC-1065,SN-38, topotecan, morpholino-doxorubicin, rhizoxin,cyanomorpholino-doxorubicin, echinomycin, combretastatin, netropsin,epothilone A and B, estramustine, cryptophysins, cemadotin, amaytansinoid, discodermolide, eleutherobin, and mitoxantrone.

Examples of other suitable agents include, but are not limited to,radionuclides, differentiation inducers, drugs, toxins, and derivativesthereof. Exemplary radionuclides include ⁹⁰Y, ¹²³I, ¹²⁵I, ¹³¹I, ¹⁸⁶Re,¹⁸⁸Re, ²¹¹At and ²¹²Bi. Exemplary drugs include methotrexate, andpyrimidine and purine analogs. Exemplary differentiation inducersinclude phorbol esters and butyric acid. Exemplary toxins include ricin,abrin, diptheria toxin, cholera toxin, gelonin, Pseudomonas exotoxin,Shigella toxin, and pokeweed antiviral protein.

In some embodiments, the therapeutic agent used in an antibody-drugconjugate is an anti-tubulin agent. Examples of anti-tubulin agentsinclude, but are not limited to, taxanes (e.g., Taxol® (paclitaxel),Taxotere® (docetaxel)), T67 (Tularik) and vinca alkyloids (e.g.,vincristine, vinblastine, vindesine, and vinorelbine). Other antitubulinagents include, for example, baccatin derivatives, taxane analogs (e.g.,epothilone A and B), nocodazole, colchicine and colcimid, estramustine,cryptophysins, cemadotin, maytansinoid, combretastatins, discodermolide,and eleutherobin.

In certain embodiments, the cytotoxic agent is a maytansinoid, anothergroup of anti-tubulin agents. For example, in specific embodiments, themaytansinoid is maytansine, DM-1 (ImmunoGen, Inc.; see also Chari etal., Cancer Res. 52:127-131 (1992)) or DM-4. In some embodiments, thetherapeutic agent is an auristatin, such as auristatin E (also known inthe art as dolastatin-10) or a derivative thereof. Typically, anauristatin E derivative is, e.g., an ester formed between auristatin Eand a keto acid. For example, auristatin E can be reacted withparaacetyl benzoic acid or benzoylvaleric acid to produce AEB and AEVB,respectively. Other typical auristatin derivatives include AFP, MMAF,and MMAE. The synthesis and structure of auristatin derivatives aredescribed in U.S. Patent Application Publication Nos. 2003-0083263,2005-0238649 and 2005-0009751; PCT Publication Nos WO 04/010957 and WO02/088172, and U.S. Pat. Nos. 6,323,315; 6,239,104; 6,034,065;5,780,588; 5,665,860; 5,663,149; 5,635,483; 5,599,902; 5,554,725;5,530,097; 5,521,284; 5,504,191; 5,410,024; 5,138,036; 5,076,973;4,986,988; 4,978,744; 4,879,278; 4,816,444; and 4,486,414.

4. CAT Nucleic Acid Molecules

Exemplary isolated CAT nucleic acid molecules of the invention consistof, consist essentially of, or comprise a nucleotide sequence thatencodes a CAT protein of the invention, an allelic variant thereof, oran ortholog or paralog thereof, for example. As used herein, an“isolated” nucleic acid molecule is one that is separated from othernucleic acid present in the natural source of the nucleic acid.Preferably, an “isolated” nucleic acid is free of sequences whichnaturally flank the nucleic acid (i.e., sequences located at the 5′ and3′ ends of the nucleic acid) in the genomic DNA of the organism fromwhich the nucleic acid is derived. However, there can be some flankingnucleotide sequences, for example up to about 5 kilobases (KB), 4 KB, 3KB, 2 KB, or 1 KB or less, particularly contiguous protein-encodingsequences and protein-encoding sequences within the same gene butseparated by introns in the genomic sequence, and flanking nucleotidesequences that contain regulatory elements. The primary consideration isthat the nucleic acid is isolated from remote and unimportant flankingsequences such that it can be subjected to the specific manipulationsdescribed herein such as recombinant expression, preparation of probesand primers, and other uses specific to the nucleic acid molecules.Moreover, an “isolated” nucleic acid molecule, such as a transcript/cDNAmolecule, can be substantially free of other cellular material, orculture medium when produced by recombinant techniques, or chemicalprecursors or other chemicals when chemically synthesized.

A nucleic acid molecule can be fused to other coding or regulatorysequences and still be considered isolated. Isolated nucleic acidmolecules can include heterologous nucleotide sequences, such asheterologous nucleotide sequences that are fused to a nucleic acidmolecule by recombinant techniques. For example, recombinant DNAmolecules contained in a vector are considered isolated. Furtherexamples of isolated DNA molecules include recombinant DNA moleculesmaintained in heterologous host cells, or purified (partially orsubstantially) DNA molecules in solution. Isolated RNA molecules includein vivo or in vitro RNA transcripts of isolated DNA molecules. Isolatednucleic acid molecules further include such molecules producedsynthetically.

Isolated nucleic acid molecules can encode a mature protein plusadditional amino or carboxyl-terminal amino acids, or amino acidsinterior to the mature protein (when the mature form has more than onepeptide chain, for instance). Such sequences may play a role inprocessing of a protein from precursor to a mature form, facilitateprotein trafficking, prolong or shorten protein half-life, or facilitatemanipulation of a protein for assay or production, among other things.As generally is the case in situ, additional amino acids may beprocessed away from the mature protein by cellular enzymes.

Isolated nucleic acid molecules include, but are not limited to,sequences encoding a CAT protein alone, sequences encoding a matureprotein with additional coding sequences (such as a leader or secretorysequence (e.g., a pre-pro or pro-protein sequence)), and sequencesencoding a mature protein (with or without additional coding sequences)plus additional non-coding sequences (e.g., introns and non-coding 5′and 3′ sequences such as transcribed but non-translated sequences thatplay a role in transcription, mRNA processing (including splicing andpolyadenylation signals), ribosome binding, and/or stability of mRNA).In addition, nucleic acid molecules can be fused to a marker sequenceencoding, for example, a peptide that facilitates purification.

Isolated nucleic acid molecules can be in the form of RNA, such as mRNA,or in the form of DNA, including cDNA and genomic DNA obtained bycloning or produced by chemical synthetic techniques or by a combinationthereof. Nucleic acid molecules, especially DNA, can be double-strandedor single-stranded. Single-stranded nucleic acid can be the codingstrand (sense strand) or the non-coding strand (anti-sense strand).

Exemplary embodiments of the invention further provide isolated nucleicacid molecules that encode fragments of a CAT protein as well as nucleicacid molecules that encode obvious variants of a CAT protein. Suchnucleic acid molecules may be naturally occurring, such as allelicvariants (same locus), paralogs (different locus), and orthologs(different organism), or can be constructed by recombinant DNA methodsor by chemical synthesis. Such non-naturally occurring variants can bemade by mutagenesis techniques, including those applied to nucleic acidmolecules, cells, or organisms. Accordingly, nucleic acid moleculevariants can contain nucleotide substitutions, deletions, inversions,and/or insertions. Variations can occur in either or both the coding andnon-coding regions, and variations can produce conservative and/ornon-conservative amino acid substitutions.

A fragment of a nucleic acid molecule typically comprises a contiguousnucleotide sequence at least 8, 10, 12, 15, 16, 18, 20, 22, 25, 30, 40,50, 100, 150, 200, 250, 500 (or any other number in-between) or morenucleotides in length. The length of a fragment can be based on itsintended use. For example, a fragment can encode epitope bearing regionsof a protein, or can be used as DNA probes and primers. Isolatedfragments can be produced by synthesizing an oligonucleotide probe usingknown techniques, for example, and can optionally be labeled and used toscreen a cDNA library, genomic DNA, or mRNA, for example. Primers can beused in PCR reactions to clone specific regions of a gene.

A probe/primer typically comprises substantially a purifiedoligonucleotide or oligonucleotide pair. An oligonucleotide typicallycomprises a nucleotide sequence that hybridizes under stringentconditions to at least about 8, 10, 12, 14, 15, 16, 17, 18, 19, 20, 21,22, 23, 24, 25, 30, 40, 50 (or any other number in-between) or morecontiguous nucleotides.

Allelic variants, orthologs, and homologs can be identified usingmethods well known in the art. These variants can comprise a nucleotidesequence encoding a protein that is typically 60-70%, 70-80%, 80-90%,90-95%, 96%, 97%, 98%, or 99% homologous to the nucleotide sequence.Such nucleic acid molecules can readily be identified as being able tohybridize under moderate to stringent conditions, to a nucleotidesequence shown in the Sequence Listing or a fragment thereof.

As used herein, the term “hybridizes under stringent conditions” isintended to describe conditions for hybridization and washing underwhich nucleotide sequences encoding a protein at least 60-70% homologousto each other typically remain hybridized to each other. The conditionscan be such that sequences at least about 60%, at least about 70%, or atleast about 80% or more homologous to each other typically remainhybridized to each other. Such stringent conditions are known to thoseskilled in the art and can be found in, for example, Current Protocolsin Molecular Biology, John Wiley & Sons, N.Y. (1989-2006), 6.3.1-6.3.6.One example of stringent hybridization conditions is hybridization in 6×sodium chloride/sodium citrate (SSC) at about 45° C., followed by one ormore washes in 0.2×SSC, 0.1% SDS at 50-65° C. Examples of moderate tolow stringency hybridization conditions are well known in the art.

Exemplary embodiments of the invention also include kits for detectingthe presence of CAT nucleic acid (e.g., DNA or mRNA) in a biologicalsample. For example, a kit can comprise reagents such as a labeled orlabelable nucleic acid and/or other agents capable of detecting CATnucleic acid in a biological sample; means for determining the amount ofCAT nucleic acid in the sample; and means for comparing the amount ofCAT nucleic acid in the sample with a standard. The nucleic acid and/orother agent can be packaged in one or more suitable containers. The kitcan further comprise instructions for using the kit to detect CATnucleic acid.

5. Vectors and Host Cells

Exemplary embodiments of the invention also provide vectors containingCAT nucleic acid molecules. The term “vector” refers to a vehicle, suchas a nucleic acid molecule, which can transport the CAT nucleic acidmolecules. When the vector is a nucleic acid molecule, the CAT nucleicacid molecules are covalently linked to the vector nucleic acid. Avector can be, for example, a plasmid, single or double stranded phage,a single or double stranded RNA or DNA viral vector, or artificialchromosome, such as a BAC, PAC, YAC, OR MAC.

A vector can be maintained in a host cell as an extrachromosomal elementwhere it replicates and produces additional copies of the CAT nucleicacid molecules. Alternatively, a vector can integrate into a host cellgenome and produce additional copies of the CAT nucleic acid moleculeswhen the host cell replicates.

Exemplary embodiments of the invention provide vectors for maintenance(cloning vectors) and vectors for expression (expression vectors) of thenucleic acid molecules, for example. Expression vectors can express aportion of, or all of, a protein sequence. Vectors can function inprokaryotic or eukaryotic cells or in both (shuttle vectors). Vectorsalso include insertion vectors, which integrate a nucleic acid moleculeinto another nucleic acid molecule, such as into the cellular genome(such as to alter in situ expression of a gene and/or gene product). Forexample, an endogenous protein-coding sequence can be entirely orpartially replaced via homologous recombination with a protein-codingsequence containing one or more specifically introduced mutations.

Expression vectors can contain cis-acting regulatory regions that areoperably-linked in the vector to the nucleic acid molecules such thattranscription of the nucleic acid molecules is allowed in a host cell.The nucleic acid molecules can be introduced into the host cell with aseparate nucleic acid molecule capable of affecting transcription. Theseparate nucleic acid molecule may provide, for example, a trans-actingfactor interacting with the cis-regulatory control region to allowtranscription of the nucleic acid molecules from the vector.Alternatively, a trans-acting factor may be supplied by a host cell.Additionally, a trans-acting factor can be produced from a vectoritself. It is understood, however, that transcription and/or translationof nucleic acid molecules can occur in cell-free systems.

Regulatory sequences to which CAT nucleic acid molecules can be operablylinked include, for example, promoters for directing mRNA transcription.These include, but are not limited to, the left promoter frombacteriophage, the lac, TRP, and TAC promoters from E. coli, the earlyand late promoters from SV40, the CMV immediate early promoter, theadenovirus early and late promoters, and retrovirus long-terminalrepeats.

In addition to control regions that promote transcription, expressionvectors can also include regions that modulate transcription, such asrepressor binding sites and enhancers. Examples include the SV40enhancer, the cytomegalovirus immediate early enhancer, polyomaenhancer, adenovirus enhancers, and retrovirus LTR enhancers.

In addition to containing sites for transcription initiation andcontrol, expression vectors can also contain sequences necessary fortranscription termination and, in the transcribed region, a ribosomebinding site for translation. Other regulatory control elements forexpression include initiation and termination codons as well aspolyadenylation signals. Numerous regulatory sequences useful inexpression vectors are well known in the art (e.g., Sambrook et al.,Molecular Cloning: A Laboratory Manual. 3rd. ed., Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y. (2001)).

A variety of expression vectors can be used to express a nucleic acidmolecule. Such vectors include chromosomal, episomal, and virus-derivedvectors, for example vectors derived from bacterial plasmids, frombacteriophage, from yeast episomes, from yeast chromosomal elements,including yeast artificial chromosomes, from viruses such asbaculoviruses, papovaviruses such as SV40, Vaccinia viruses,adenoviruses, poxviruses, pseudorabies viruses, and retroviruses.Vectors may also be derived from combinations of these sources such asthose derived from plasmid and bacteriophage genetic elements, e.g.cosmids and phagemids. Appropriate cloning and expression vectors forprokaryotic and eukaryotic hosts are described in Sambrook et al.,Molecular Cloning: A Laboratory Manual. 3rd. ed., Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y. (2001).

A regulatory sequence can provide constitutive expression in one or morehost cells (e.g., tissue specific) or can provide for inducibleexpression in one or more cell types such as by temperature, nutrientadditive, or exogenous factors such as a hormone or other ligand. Avariety of vectors providing for constitutive and inducible expressionin prokaryotic and eukaryotic hosts are well known in the art.

Nucleic acid molecules can be inserted into vector nucleic acid bywell-known methodology. For example, the DNA sequence that willultimately be expressed can be joined to an expression vector bycleaving the DNA sequence and the expression vector with one or morerestriction enzymes and then ligating the fragments together. Proceduresfor restriction enzyme digestion and ligation are well known in the art.

A vector containing a nucleic acid molecule of interest can beintroduced into an appropriate host cell for propagation or expressionusing well-known techniques. Bacterial cells include, but are notlimited to, E. coli, Streptomyces, and Salmonella typhimurium.Eukaryotic cells include, but are not limited to, yeast, insect cellssuch as Drosophila, animal cells such as COS and CHO cells (e.g., DG44or CHO-s), and plant cells.

As described herein, it may be desirable to express a protein as afusion protein. Accordingly, exemplary embodiments of the inventionprovide fusion vectors that allow for the production of fusion proteins.Fusion vectors can, for example, increase the expression of arecombinant protein; increase the solubility of a recombinant protein,and/or aid in the purification of a protein such as by acting as aligand for affinity purification. A proteolytic cleavage site can beintroduced at the junction of the fusion moiety so that the desiredprotein can ultimately be separated from the fusion moiety. Proteolyticenzymes include, but are not limited to, factor Xa, thrombin, andenteroenzyme. Typical fusion expression vectors include pGEX (Smith etal., Gene 67:31-40 (1988)), pMAL (New England Biolabs, Beverly, Mass.)and pRIT5 (Pharmacia, Piscataway, N.J.), which fuse glutathioneS-transferase (GST), maltose E binding protein, or protein A,respectively, to a target recombinant protein. Examples of suitableinducible non-fusion E. coli expression vectors include pTrc (Amann etal., Gene 69:301-315 (1988)) and pET 11d (Studier et al., GeneExpression Technology: Methods in Enzymology 185:60-89 (1990)).

Recombinant protein expression can be maximized in host bacteria byproviding a genetic background wherein the host cell has an impairedcapacity to proteolytically cleave the recombinant protein (Gottesman,S., Gene Expression Technology: Methods in Enzymology 185, AcademicPress, San Diego, Calif. (1990), pp. 119-128). Alternatively, thesequence of a nucleic acid molecule of interest can be altered toprovide preferential codon usage for a specific host cell, such as E.coli (Wada et al., Nucleic Acids Res. 20:2111-2118 (1992)).

CAT nucleic acid molecules can, for example, be expressed by expressionvectors in a yeast host. Examples of vectors for expression in yeast(e.g., S. cerevisiae) include pYepSec1 (Baldari, et al., EMBO J.6:229-234 (1987)), pMFa (Kurjan et al., Cell 30:933-943 (1982)), pJRY88(Schultz et al., Gene 54:113-123 (1987)), and pYES2 (InvitrogenCorporation, San Diego, Calif.). Nucleic acid molecules can also beexpressed in insect cells using, for example, baculovirus expressionvectors. Baculovirus vectors available for expression of proteins incultured insect cells (e.g., Sf 9 cells) include the pAc series (Smithet al., Mol. Cell Biol. 3:2156-2165 (1983)) and the pVL series (Lucklowet al., Virology 170:31-39 (1989)). Nucleic acid molecules can also beexpressed in mammalian cells using mammalian expression vectors.Examples of mammalian expression vectors include pCDM8 (Seed, B. Nature329:840 (1987)), pMT2PC (Kaufman et al., EMBO J. 6:187-195 (1987)), andCHEF (U.S. Pat. No. 5,888,809).

The expression vectors listed herein are provided by way of example onlyof well-known vectors available to those of ordinary skill in the artthat would be useful to express CAT nucleic acid molecules. The personof ordinary skill in the art would be aware of other vectors suitablefor maintenance, propagation, and/or expression of CAT nucleic acidmolecules (e.g., Sambrook et al. Molecular Cloning: A Laboratory Manual.3rd. ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.(2001).

Exemplary embodiments of the invention also encompasses vectors in whichCAT nucleic acid molecules are cloned into a vector in reverseorientation, but operably linked to a regulatory sequence that permitstranscription of antisense RNA. Thus, an antisense transcript can beproduced to all, or to a portion, of a CAT nucleic acid molecule,including coding and non-coding regions. Expression of this antisenseRNA may be subject to each of the parameters described above in relationto expression of the sense RNA (e.g., regulatory sequences, constitutiveor inducible expression, tissue-specific expression).

Exemplary embodiments of the invention provide recombinant host cellscontaining the vectors described herein. Host cells include, forexample, prokaryotic cells, lower eukaryotic cells such as yeast, othereukaryotic cells such as insect cells, and higher eukaryotic cells suchas mammalian cells.

Recombinant host cells can be prepared by introducing vector constructs,such as described herein, into cells by techniques readily available toa person of ordinary skill in the art. These techniques include, but arenot limited to, calcium phosphate transfection, DEAE-dextran-mediatedtransfection, cationic lipid-mediated transfection, electroporation,transduction, infection, lipofection, microinjection, and othertechniques such as those found in Sambrook, et al. (Molecular Cloning: ALaboratory Manual. 3rd. ed., Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y. (2001).

For example, using techniques such as these, a retroviral or other viralvector can be introduced into mammalian cells. Examples of mammaliancells into which a retroviral vector can be introduced include, but arenot limited to, primary mammalian cultures or continuous mammaliancultures, COS cells, NIH3T3, 293 cells (ATCC #CRL 1573), and dendriticcells.

Host cells can contain more than one vector. Thus, different nucleotidesequences can be introduced on different vectors of the same cell.Similarly, nucleic acid molecules of interest can be introduced eitheralone or with other unrelated nucleic acid molecules such as thoseproviding trans-acting factors for expression vectors. When more thanone vector is introduced into a cell, the vectors can be introducedindependently, co-introduced, or joined to the nucleic acid moleculevector.

Bacteriophage and viral vectors can be introduced into cells as packagedor encapsulated virus by standard procedures for infection andtransduction. Viral vectors can be replication-competent orreplication-defective. If viral replication is defective, replicationcan occur in host cells that provide functions that complement thedefects.

Vectors can include selectable markers that enable the selection of asubpopulation of cells that contain the recombinant vector constructs.Markers can be contained in the same vector that contains the nucleicacid molecules of interest or can be on a separate vector. Exemplarymarkers include tetracycline or ampicillin-resistance genes forprokaryotic host cells, and dihydrofolate reductase or neomycinresistance for eukaryotic host cells. However, any marker that providesselection for a phenotypic trait can be used.

While mature proteins can be produced in bacteria, yeast, mammaliancells, and other cells under the control of appropriate regulatorysequences, cell-free transcription and translation systems can also beused to produce these proteins using RNA derived from the DNA constructsdescribed herein.

If secretion of a protein is desired, appropriate secretion signals canbe incorporated into a vector. The signal sequence can be endogenous orheterologous to the protein.

If a protein is not secreted into a medium, the protein can be isolatedfrom a host cell by standard disruption procedures, includingfreeze/thaw, sonication, mechanical disruption, use of lysing agents,and the like. A protein can then be recovered and purified by well-knownpurification methods including, for example, ammonium sulfateprecipitation, acid extraction, anion or cationic exchangechromatography, phosphocellulose chromatography, hydrophobic-interactionchromatography, affinity chromatography, hydroxylapatite chromatography,lectin chromatography, or high performance liquid chromatography.

It is also understood that, depending upon the host cell used inrecombinant production of a protein, proteins can have variousglycosylation patterns or can be non-glycosylated, such as when producedin bacteria. In addition, proteins can include an initial modifiedmethionine in some instances as a result of a host-mediated process.

Recombinant host cells that express a CAT protein have a variety ofuses. For example, such host cells are useful for producing CATproteins, which can be further purified to produce desired amounts ofthe protein or fragments thereof. Thus, host cells containing expressionvectors are useful for protein production.

Host cells are also useful for conducting cell-based assays involving aCAT protein or fragments thereof. For example, a recombinant host cellexpressing a CAT protein can be used to assay compounds that stimulateor inhibit the protein's function.

Host cells are also useful for identifying mutant CAT proteins in whichthe protein's function is affected. Host cells expressing mutantproteins are useful for assaying compounds that have a desired effect onthe mutant proteins (e.g., stimulating or inhibiting function),particularly if the mutant proteins naturally occur and give rise to apathology.

6. Diagnosis and Treatment in General

The following terms, as used in the present specification and claims,are intended to have the meaning as defined below, unless indicatedotherwise.

As used herein, a “biological sample” (or just “sample”) can comprise,for example, tissue, blood, sera, cells, cell lines, or biologicalfluids such as plasma, interstitial fluid, urine, cerebrospinal fluid,and the like. A biological sample is typically, although notnecessarily, obtained from an individual by a medical practitioner.

As used herein, a “subject” can be a mammalian subject or non-mammaliansubject, preferably a mammalian subject. A mammalian subject can be ahuman or non-human, preferably a human. The terms “subject”,“individual”, and “patient” are used herein interchangeably.

A “healthy” or “normal” subject or biological sample is a subject orbiological sample in which the disease of interest is not detectable, asascertained by using conventional diagnostic methods (such a biologicalsample can interchangeably be referred to as a “control” sample).

As used herein, “disease(s)” include cancer, particularly the cancersidentified in Table 1 (as indicated for each peptide) and/or Table 3,and associated diseases and pathologies.

The terms “diagnose” (or “diagnosing”, etc.) and “assess” (or“assessing”, etc.) are used herein interchangeably. Diagnosing orassessing diseases can include, for example, initially detecting thepresence of a disease; determining a specific stage, sub-type, or otherclassification or characteristic of a disease; prognosing the futurecourse of a disease; monitoring disease progression (e.g., monitoringmetastatic spread of a cancer) or remission; determining or predictingresponse to a treatment; determining or predicting recurrence of adisease; and/or determining the likelihood of developing a disease inthe future.

“Treat”, “treating”, or “treatment” of a disease includes: (1)inhibiting the disease, i.e., arresting or reducing the development ofthe disease or its clinical symptoms, or (2) relieving the disease,i.e., causing regression of the disease or its clinical symptom(s).

The term “prophylaxis” is used to distinguish from “treatment,” and toencompass both “preventing” and “suppressing.” It is not always possibleto distinguish between “preventing” and “suppressing,” as the ultimateinductive event or events may be unknown, latent, or the patient is notascertained until well after the occurrence of the event or events.Therefore, the term “protection”, as used herein, is meant to include“prophylaxis.”

A “therapeutically effective amount” means the amount of an agent that,when administered to a subject for treating a disease, is sufficient toeffect such treatment for the disease. The “therapeutically effectiveamount” can vary depending on such factors as the agent, the disease andits severity, and the age, weight, etc., of the subject to be treated.

A “differential level” is a level of a target (e.g., CAT protein ornucleic acid) in a test sample (e.g., disease sample, or drug resistantcells) either above or below the level of the same target in acorresponding control or normal sample (e.g., a control cell line or abiological sample from a healthy individual, or cellsresponsive/sensitive to a drug).

Exemplary embodiments of the invention provide methods for treatingdiseases, especially cancer, particularly the cancers identified inTable 1 (as indicated for each peptide) and/or Table 3, comprisingadministering to a patient a therapeutically effective amount of anantagonist, agonist, or a pharmaceutical composition thereof. Exemplaryembodiments of the invention further provide agonists and antagonists toCAT proteins, as well as pharmaceutical compositions that comprise anagonist or antagonist with a suitable carrier such as a pharmaceuticallyacceptable excipient.

Exemplary agonists or antagonists include antibodies that specificallybind to a CAT protein. Antibodies can be used alone or in combinationwith one or more other therapeutic agents (e.g., as an antibody-drugconjugate or a combination therapy). Further examples of molecules thatcan be used as antagonists include, but are not limited to, smallmolecules that inhibit the function or abundance level of CAT, andinhibitory nucleic acid molecules such as RNAi or antisense nucleic acidmolecules that specifically hybridize to CAT nucleic acid.

Exemplary embodiments of the invention further encompass novel agentsidentified by screening assays using CAT, such as the screening assaysdescribed herein, as well as methods of using these agents, such as fortreatment or diagnostic purposes. For example, an agent identified asdescribed herein (e.g., a CAT-modulating agent, a CAT-specific nucleicacid molecule such as an RNAi or antisense molecule, a CAT-specificantibody, a CAT-specific antibody-drug conjugate, or a CAT-bindingpartner) can be used in an animal or other model, such as to determineefficacy, toxicity, or side effects of treatment with the agent.

Modulators of CAT protein activity, such as modulators identifiedaccording to the drug screening assays described herein, can be used totreat a subject with a disorder mediated by a CAT, e.g., by treatingcells or tissues that express CAT at a differential level. Methods oftreatment can include the step of administering a modulator of CATactivity in a pharmaceutical composition to a subject in need of suchtreatment.

In certain exemplary embodiments, if decreased expression or activity ofa protein is desired, an antibody to the protein or aninhibitor/antagonist and the like, or a pharmaceutical agent containingone or more of these molecules, can be administered to an individual. Inother exemplary embodiments, if increased expression or activity of aprotein is desired, the protein itself or an agonist/enhancer and thelike, or a pharmaceutical agent containing one or more of thesemolecules, can be administered. Administration can be effected bymethods well known in the art and may include delivery by an antibodyspecifically targeted to the protein. Neutralizing antibodies, whichinhibit dimer formation, can be used when decreased expression oractivity of a protein is desired.

Although modulating agents can be administered in a pure orsubstantially pure form, modulating agents can also be administed aspharmaceutical compositions, formulations, or preparations with acarrier. Exemplary formulations of the invention, such as for human orveterinary use, comprise a suitable active CAT-modulating agent,together with one or more pharmaceutically acceptable carriers and,optionally, other therapeutic ingredients. The carrier(s) are“acceptable” in the sense of being compatible with other ingredients ofa formulation and not deleterious to the recipient thereof. Theformulations can be presented in unit dosage form and can be prepared byany method known to the skilled artisan.

Examples of suitable pharmaceutical carriers include proteins such asalbumins (e.g., U.S. Pat. No. 4,507,234), peptides and polysaccharidessuch as aminodextran (e.g., U.S. Pat. No. 4,699,784), and water. Acarrier can also bear an agent by noncovalent bonding or byencapsulation, such as within a liposome vesicle (e.g., U.S. Pat. Nos.4,429,008 and 4,873,088). Carriers specific for radionuclide agentsinclude radiohalogenated small molecules and chelating compounds. Forexample, U.S. Pat. No. 4,735,792 discloses representativeradiohalogenated small molecules and their synthesis. A radionuclidechelate can be formed from chelating compounds that include thosecontaining nitrogen and sulfur atoms as the donor atoms for binding themetal, metal oxide, radionuclide. For example, U.S. Pat. No. 4,673,562discloses representative chelating compounds and their synthesis.

Methods of preparing pharmaceutical formulations typically include thestep of bringing into association the active ingredient with thecarrier, which constitutes one or more accessory ingredients.Formulations can be prepared by uniformly and intimately bringing intoassociation the active ingredient with liquid carriers or finely dividedsolid carriers, or both, and then, if necessary, shaping the productinto the desired formulation.

Formulations suitable for intravenous, intramuscular, subcutaneous, orintraperitoneal administration can comprise sterile aqueous solutions ofthe active ingredient with solutions, which can be isotonic with theblood of the recipient. Such formulations can be prepared by dissolvingsolid active ingredient in water containing physiologically compatiblesubstances such as sodium chloride (e.g., 0.1-2.0 M), glycine, and thelike, and having a buffered pH compatible with physiological conditionsto produce an aqueous solution, and rendering the solution sterile.These may be present in unit or multi-dose containers, for example,sealed ampoules or vials.

Exemplary formulations of the invention can incorporate a stabilizer.Exemplary stabilizers include polyethylene glycol, proteins,saccharides, amino acids, inorganic acids, detergents, and organicacids, which can be used either alone or as admixtures. Thesestabilizers can be incorporated in an amount of, for example,0.11-10,000 parts by weight per part by weight of an agent. If two ormore stabilizers are to be used, their total amount can be within therange specified above. These stabilizers can be used in aqueoussolutions at an appropriate concentration and pH. The specific osmoticpressure of such aqueous solutions can be in the range of 0.1-3.0osmoles, preferably in the range of 0.8-1.2. The pH of the aqueoussolution can be adjusted to be within the range of 5.0-9.0, preferablywithin the range of 6-8. In formulating an antibody or antibody-drugconjugate, an anti-adsorption agent can be used.

Additional pharmaceutical methods can be employed to control duration ofaction. Controlled release can be achieved through the use of polymer tocomplex or absorb the proteins or their derivatives. Controlled deliverycan be achieved by selecting appropriate macromolecules (e.g.,polyester, polyamino acids, polyvinyl, pyrrolidone,ethylenevinylacetate, methylcellulose, carboxymethylcellulose, orprotamine sulfate) and the concentration of macromolecules as well asthe methods of incorporation in order to control release. Anotherpossible method to control the duration of action by controlled-releasepreparations is to incorporate an anti-CAT antibody into particles of apolymeric material such as polyesters, polyamino acids, hydrogels,poly(lactic acid) or ethylene vinylacetate copolymers. Alternatively,instead of incorporating these agents into polymeric particles, it ispossible to entrap these materials in microcapsules prepared, forexample, by coacervation techniques or by interfacial polymerization,for example, hydroxymethylcellulose or gelatin-microcapsules andpoly(methylmethacylate) microcapsules, respectively, or in colloidaldrug delivery systems, for example, liposomes, albumin microspheres,microemulsions, nanoparticles, and nanocapsules or in macroemulsions.

When oral preparations are desired, the compositions can be combinedwith typical carriers, such as lactose, sucrose, starch, talc magnesiumstearate, crystalline cellulose, methyl cellulose, carboxymethylcellulose, glycerin, sodium alginate or gum arabic, among others.

Any of the therapeutic agents provided herein may be administered incombination with other therapeutic agents. Selection of agents for usein combination therapy can be made by one of ordinary skill in the artaccording to conventional pharmaceutical principles. A combination oftherapeutic agents may act synergistically to affect treatment of aparticular disorder at a lower dosage of each agent.

7. Methods of Detection and Diagnosis Based on CAT Proteins

CAT proteins are useful for diagnosing a disease, or predisposition to adisease, particularly diseases in which the protein is over- orunder-expressed, especially cancer, particularly the cancers identifiedin Table 1 (as indicated for each peptide) and/or Table 3. Thediagnostic methods may be further suitable for monitoring diseaseprogression in patients undergoing treatment, or for testing forreoccurrence of disease in patients who were previously treated for adisease, for example. Accordingly, exemplary embodiments of theinvention provide methods for detecting the presence of, or abundancelevels of, a CAT protein in a biological sample.

In vitro techniques for detection of proteins include, but are notlimited to, enzyme linked immunosorbent assays (ELISAs), Western blots,immunoprecipitations, and immunofluorescence using a detection reagent,such as an antibody or protein binding agent. Alternatively, a proteincan be detected in vivo in a subject by introducing into the subject alabeled antibody (or other types of detection agent) specific for theprotein target. For example, an antibody can be labeled with aradioactive marker whose presence and location in a subject can bedetected by standard imaging techniques. Particularly useful are methodsthat detect variants of a protein (e.g., allelic variants or mutations)and methods that detect fragments of a protein in a sample.

Proteins can be isolated from a biological sample (such as from apatient having a disease) and assayed for the presence of a mutation. Amutation can include, for example, one or more amino acid substitutions,deletions, insertions, rearrangements (such as from aberrant splicingevents), or inappropriate post-translational modifications. Examples ofanalytic methods useful for detecting mutations in a protein include,but are not limited to, altered electrophoretic mobility, alteredtryptic peptide digest, altered protein activity in cell-based orcell-free assays, alteration in substrate or antibody-binding patterns,altered isoelectric point, and direct amino acid sequencing.

Information obtained by detecting a protein can be used, for example, todetermine prognosis and appropriate course of treatment for a disease.For example, individuals with a particular CAT expression level or stageof disease may respond differently to a given treatment that individualslacking CAT expression, or individuals over- or under-expressing CAT.Information obtained from diagnostic methods of the invention canprovide for the personalization of diagnosis and treatment.

In exemplary embodiments, the invention provides methods for diagnosingdisease (including, for example, monitoring treatment response orrecurrence of disease following treatment) in a subject comprising:determining the abundance level of CAT (e.g., CAT protein or nucleicacid, or protein or nucleic acid fragments thereof) in a test samplefrom the subject; wherein a difference in the abundance level of CATrelative to the abundance level of CAT in a test sample from a healthysubject, or the level established for a healthy subject, is indicativeof disease.

Exemplary embodiments of the invention provide methods for diagnosingdiseases having differential protein expression. For example, normal,control, or standard values (e.g., that represent typical expressionlevels of a protein in healthy subjects) can be established, such as bycombining body fluids, tissues, or cell extracts taken from a normalhealthy mammalian or human subject with specific antibodies to a proteinunder conditions for complex formation. Standard values for complexformation in normal and disease tissues can be established by variousmethods, such as photometric means. Complex formation, as it isexpressed in a test sample, can be compared with the standard values.Deviation from a normal standard and toward a disease standard canprovide parameters for disease diagnosis or prognosis while deviationaway from a disease standard and toward a normal standard can be used toevaluate treatment efficacy, for example.

Immunological methods for detecting and measuring complex formation as ameasure of protein expression using either specific polyclonal ormonoclonal antibodies are known in the art. Examples of such techniquesinclude ELISAs, radioimmunoassays (RIAs), flow cytometry (also referredto as fluorescence-activated cell sorting, or FACS), and antibodyarrays. Such immunoassays typically involve the measurement of complexformation between a protein and its specific antibody. These assays andtheir quantitation against purified, labeled standards are well known inthe art (Ausubel, supra, unit 10.1-10.6). For example, a two-site,monoclonal-based immunoassay utilizing antibodies reactive to twonon-interfering epitopes can be utilized, and competitive binding assaycan also be utilized (Pound (1998) Immunochemical Protocols, HumanaPress, Totowa N.J.).

For diagnostic applications, an antibody can be labeled with adetectable moiety (interchangeably referred to as a “label” or“detectable substance”), such as to facilitate detection by variousimaging methods. Methods for detection of labels include, but are notlimited to, fluorescence, light, confocal, and electron microscopy;magnetic resonance imaging and spectroscopy; fluoroscopy, computedtomography and positron emission tomography. Examples of suitable labelsinclude, but are not limited to, fluorescein, rhodamine, eosin and otherfluorophores, radioisotopes, gold, gadolinium and other lanthanides,paramagnetic iron, fluorine-18 and other positron-emittingradionuclides. Additionally, labels may be bi- or multi-functional andbe detectable by more than one of the methods listed. Antibodies may bedirectly or indirectly labeled. Attachment of labels to antibodiesincludes covalent attachment of a label, incorporation of a label intoan antibody, and covalent attachment of a chelating compound for bindingof a label, among others well known in the art.

Numerous detectable moieties are available for labeling antibodies,including, but not limited to, those in the following categories:

(a) Radioisotopes, such as ³⁶S, ¹⁴C, ¹²⁵I, ³H, and ¹³¹I. An antibody canbe labeled with a radioisotope using the techniques described in CurrentProtocols in Immunology, vol 1-2, Coligen et al., Ed.,Wiley-Interscience, New York, Pubs. (1991-2006), for example, andradioactivity can be measured using scintillation counting.

(b) Fluorescent labels such as rare earth chelates (europium chelates)or fluorescein and its derivatives, rhodamine and its derivatives,dansyl, Lissamine, phycoerythrin and Texas Red are available.Fluorescent labels can be conjugated to an antibody using the techniquesdisclosed in Current Protocols in Immunology, supra, for example.Fluorescence can be quantified using a fluorometer.

(c) Various enzyme-substrate labels are available (e.g., U.S. Pat. Nos.4,275,149 and 4,318,980). An enzyme generally catalyzes a chemicalalteration of a chromogenic substrate which can be measured usingvarious techniques. For example, an enzyme may catalyze a color changein a substrate, which can be measured spectrophotometrically.Alternatively, an enzyme may alter the fluorescence or chemiluminescenceof a substrate. Techniques for quantifying a change in fluorescence aredescribed herein and well known in the art A chemiluminescent substratebecomes electronically excited by a chemical reaction and may then emitlight which can be measured (using a chemiluminometer, for example) ordonates energy to a fluorescent acceptor. Examples of enzymatic labelsinclude luciferases (e.g., firefly luciferase and bacterial luciferase;U.S. Pat. No. 4,737,456), luciferin, 2,3-dihydrophthalazinediones,malate dehydrogenase, urease, peroxidase such as horseradish peroxidase(HRPO), alkaline phosphatase, β-galactosidase, glucoamylase, lysozyme,saccharide oxidases (e.g., glucose oxidase, galactose oxidase, andglucose-6-phosphate dehydrogenase), heterocyclic oxidases (such asuricase and xanthine oxidase), lactoperoxidase, microperoxidase, and thelike. Techniques for conjugating enzymes to antibodies are described inO'Sullivan et al., Methods for the Preparation of Enzyme-AntibodyConjugates for Use in Enzyme Immunoassay, in Methods in Enzyme. (Ed. J.Langone & H. Van Vunakis), Academic press, New York, 73: 147-166 (1981).

A label can be indirectly conjugated with an antibody. The skilledartisan will be aware of various techniques for achieving this. Forexample, an antibody can be conjugated with biotin and any of the threebroad categories of labels mentioned above can be conjugated withavidin, or vice versa. Biotin binds selectively to avidin and thus, thelabel can be conjugated with the antibody in this indirect manner.Alternatively, to achieve indirect conjugation of a label with anantibody, an antibody can be conjugated with a small hapten (e.g.,digoxin) and one of the different types of labels mentioned above can beconjugated with an anti-hapten antibody (e.g., anti-digoxin antibody).Thus, indirect conjugation of a label with an antibody can be achieved.

Antibodies can be used to isolate CAT proteins by standard techniques,such as affinity chromatography or immunoprecipitation, and antibodiescan facilitate the purification of the natural protein from cells andrecombinantly-produced protein expressed in host cells. Biologicalsamples can be tested directly for the presence of a CAT protein byassays (e.g., ELISA or radioimmunoassay) and format (e.g., microwells,dipstick, etc., as described in International Patent Publication WO93/03367). Alternatively, proteins in a sample can be size separated(e.g., by polyacrylamide gel electrophoresis (PAGE)), in the presence orabsence of sodium dodecyl sulfate (SDS), and the presence of a CATdetected by immunoblotting (e.g., Western blotting).

Antibody binding can also be detected by “sandwich” immunoassays,immunoradiometric assays, gel diffusion precipitation reactions,immunodiffusion assays, in situ immunoassays (e.g., using colloidalgold, enzyme or radioisotope labels, for example), precipitationreactions, agglutination assays (e.g., gel agglutination assays,hemagglutination assays, etc.), complement fixation assays,immunofluorescence assays, protein A assays, and immunoelectrophoresisassays, etc.

In certain exemplary embodiments, antibody binding can be detected bydetecting a label on the primary antibody. In other exemplaryembodiments, a primary antibody can be detected by detecting binding ofa secondary antibody or reagent to the primary antibody. In furtherexemplary embodiments, the secondary antibody is labeled. Numerous meansare known in the art for detecting binding in an immunoassay and arewithin the scope of the invention. In some embodiments, an automateddetection assay is utilized. Methods for the automation of immunoassaysare well known in the art (e.g., U.S. Pat. Nos. 5,885,530: 4,981,785:6,159,750: and 5,358,691, each of which is herein incorporated byreference). In some embodiments, the analysis and presentation ofresults are also automated. For example, in some embodiments, softwarethat generates a prognosis based on the presence or absence of one ormore antigens can be implemented.

Competitive binding assays typically rely on the ability of a labeledstandard to compete with a test sample for binding with a limited amountof antibody. The amount of antigen in the test sample is inverselyproportional to the amount of standard that becomes bound to theantibodies. To facilitate determining the amount of standard thatbecomes bound, the antibodies generally are insolubilized before orafter the competition. As a result, the standard and test sample thatare bound to the antibodies can be separated from the standard and testsample that remain unbound.

Sandwich assays typically involve the use of two antibodies, eachcapable of binding to a different immunogenic portion, or epitope, ofthe protein to be detected. In typical sandwich assays, the test sampleto be analyzed is bound by a first antibody, which is immobilized on asolid support, and thereafter a second antibody binds to the testsample, thus forming an insoluble three-part complex (e.g., U.S. Pat.No. 4,376,110). The second antibody can itself be labeled with adetectable moiety (direct sandwich assays) or can be measured using ananti-immunoglobulin antibody that is labeled with a detectable moiety(indirect sandwich assay). For example, one type of sandwich assay is anELISA assay, in which case the detectable moiety is an enzyme.

Antibodies can also be used for in vivo diagnostic assays. Generally, anantibody can be labeled with a radionuclide (such as ¹¹¹In, ⁹⁹Tc, ¹⁴C,¹³¹I, ³H, ³²P or ³⁵S) so that disease cells or tissues can be localizedusing immunoscintiography, for example. In certain embodiment,antibodies or fragments thereof bind to the extracellular domains of twoor more CAT proteins and the affinity value (Kd) is less than 1×10⁸ M.

For immunohistochemistry, a disease tissue sample may be, for example,fresh or frozen or may be embedded in paraffin and fixed with apreservative such as formalin. A fixed or embedded section can becontacted with a labeled primary antibody and secondary antibody,wherein the antibody is used to detect CAT protein expression in situ.

Antibodies can be used to detect a target protein in situ, in vitro, orin a cell lysate or supernatant in order to evaluate the abundance andpattern of expression. Also, antibodies can be used to assess abnormaltissue distribution or abnormal expression during development orprogression of a biological condition. Antibodies against CAT proteinsare useful for detecting the presence of the proteins in cells ortissues to determine the pattern of expression of the proteins amongvarious tissues in an organism and over the course of the organism'sdevelopment.

Further, antibodies can be used to assess expression in disease statessuch as in active stages of a disease or in an individual with apredisposition toward disease related to the protein's function. When adisorder is caused by inappropriate tissue distribution, developmentalexpression, or level of expression of a protein, or expressed/processedform, for example, an antibody can be prepared against the normalprotein. If a disorder is characterized by a specific mutation in aprotein, antibodies specific for the mutant protein can be used to assayfor the presence of the specific mutant protein and to target the mutantprotein for therapeutic purposes. Antibodies are also useful asdiagnostic tools, as immunological markers for aberrant protein analyzedby electrophoretic mobility, isoelectric point, tryptic peptide digest,and other physical assays known in the art.

Certain exemplary diagnostic methods of the invention can also includemonitoring a treatment modality. Accordingly, where treatment isultimately aimed at correcting, for example, the function, activity,expression level, tissue distribution, or developmental expression of aprotein, antibodies directed against the protein can be used to monitortherapeutic efficacy and to modify a treatment regimen as necessary.

Additionally, antibodies to a target protein are useful inpharmacogenomic analysis. For example, antibodies prepared againstpolymorphic proteins can be used to identify individuals that requiremodified treatment modalities. Moreover, the target proteins andantibodies thereto can be used for clinical trials, such as to identifyindividuals that should be included (e.g., individuals more likely torespond to a therapy) or excluded (e.g., individuals less likely torespond to a therapy, or individuals more likely to experience harmfulside effects from a therapy) from a clinical trial.

The invention also encompasses kits for using antibodies to detect thepresence of a target protein in a biological sample. An exemplary kitcan comprise antibodies such as a labeled or labelable antibody and acompound or agent for detecting protein in a biological sample; meansfor determining the amount of protein in the sample; means for comparingthe amount of protein in the sample with a standard; and instructionsfor use. Such a kit can be configured to detect a single target proteinor epitope or can be configured to detect one of a multitude ofepitopes, such as in an antibody detection array.

LC/MS and ICAT

In certain exemplary embodiments, the invention provides detection ordiagnostic methods of a CAT by using LC/MS. Proteins can be preparedfrom cells by methods known in the art (e.g., Zhang et al., NatureBiotechnology 21(6):660-666 (2003)). The differential expression ofproteins in disease and healthy (or drug-resistant and drug-sensitive,for example) samples can be quantitated using mass spectrometry and ICAT(Isotope Coded Affinity Tag) labeling, which is known in the art. ICATis an isotope label technique that allows for discrimination between twopopulations of proteins, such as a healthy and a disease sample.Over-expression or under-expression of a CAT protein, as measured byICAT, can indicate, for example, the likelihood of having or developinga disease or an associated pathology.

LC/MS spectra can be collected for labeled samples and processed asfollows. The raw scans from the LC/MS instrument can be subjected topeak detection and noise reduction software. Filtered peak lists canthen be used to detect ‘features’ corresponding to specific peptidesfrom the original sample(s). Features are characterized by theirmass/charge ratio, charge, retention time, isotope pattern, and/orintensity, for example.

The intensity of a peptide present in both healthy and disease samplescan be used to calculate the differential expression, or relativeabundance, of the peptide. The intensity of a peptide found exclusivelyin one sample can be used to calculate a theoretical expression ratiofor that peptide (singleton). Expression ratios can be calculated foreach peptide in an assay or experiment.

Statistical tests can be performed to assess the robustness of the dataand select statistically significant differentials. To ensure theaccuracy of data, the following steps can be taken: a) ensure thatsimilar features are detected in all replicates of an experiment; b)assess the distribution of the log ratios of all peptides (a Gaussian isexpected); c) calculate the overall pair wise correlations between ICATLC/MS maps to ensure that the expression ratios for peptides arereproducible across multiple replicates; and d) aggregate multipleexperiments in order to compare the expression ratio of a peptide inmultiple diseases or disease samples.

8. Methods of Treatment Based on CAT Proteins

a. Antibody Therapy

Antibodies of the invention can be used for therapeutic purposes. It iscontemplated that antibodies of the invention may be used to treat amammal, preferably a human, with a disease, especially cancer,particularly the cancers identified in Table 1 (as indicated for eachpeptide) and/or Table 3. The antibodies can be delivered alone, in apharmaceutical composition (such as with a carrier), or conjugated toone or more therapeutic agents, for example.

Antibodies can be useful for modulating (e.g., agonizing orantagonizing) protein function, such as for therapeutic purposes.Antibodies can also be useful for inhibiting protein function by, forexample, blocking the binding of a CAT protein to a binding partner suchas a substrate, which can be useful therapeutically. Antibodies can beprepared against, for example, specific portions of a protein thatcontain domains required for protein function, or against intact proteinthat is associated with a cell membrane.

Antibodies of the invention can also be used for enhancing the immuneresponse. The antibodies can be administered in amounts similar to thoseused for other therapeutic administrations of antibodies. For example,pooled gamma globulin can be administered at a range of about 1 mg toabout 100 mg per patient.

Antibodies reactive with CAT proteins can be administered alone or inconjunction with other therapies, such as anti-cancer therapies, to amammal afflicted with cancer or other disease. Examples of anti-cancertherapies include, but are not limited to, chemotherapy, radiationtherapy, and adoptive immunotherapy therapy with TIL (tumor infiltratinglymphocytes).

The selection of an antibody subclass for therapy may depend upon thenature of the antigen to be acted upon. For example, an IgM may bepreferred in situations where the antigen is highly specific for thediseased target and rarely occurs on normal cells. However, where thedisease-associated antigen is also expressed in normal tissues, althoughat lower levels, the IgG subclass may be preferred. The IgG subclass maybe preferred in these instances because the binding of at least two IgGmolecules in close proximity is typically required to activatecomplement, and therefore less complement-mediated damage may occur innormal tissues that express smaller amounts of the antigen and thus bindfewer IgG antibody molecules. Furthermore, IgG molecules, by beingsmaller, may be more able than IgM molecules to localize to a diseasedtissue.

A mechanism for antibody therapy can be that a therapeutic antibodyrecognizes a cell surface, secreted, or cytosolic target protein that isexpressed (preferably, over-expressed) in a disease cell. By NK cell orcomplement activation, or conjugation of the antibody with animmunotoxin or radiolabel, the interaction of the antibody with thetarget protein can abrogate ligand/receptor interaction or activation ofapoptosis, for example.

Potential mechanisms of antibody-mediated cytotoxicity of diseased cellsinclude phagocyte (antibody-dependent cellular cytotoxicity (ADCC)),complement (complement-dependent cytotoxicity (CDC)), naked antibody(receptor cross-linking apoptosis and growth factor inhibition), ortargeted payload labeled with a therapeutic agent, such as aradionuclide, immunotoxin, or immunochemotherapeutic or othertherapeutic agent.

In certain exemplary embodiments, an antibody is administered to anonhuman mammal for the purposes of obtaining preclinical data, forexample. Exemplary nonhuman mammals to be treated include nonhumanprimates, dogs, cats, rodents, and other mammals in which preclinicalstudies are performed. Such mammals may be established animal models fora disease or may be used to study toxicity of an antibody of interest,for example. Dose escalation studies may be performed in the mammal, forexample.

An antibody can be administered to an individual by any suitable means,including parenteral, subcutaneous, intraperitoneal, intrapulmonary, andintranasal, and, if desired for local immunomodulatory treatment,intralesional administration. Parenteral infusions includeintramuscular, intravenous, intraarterial, intraperitoneal, orsubcutaneous administration. In addition, an antibody can beadministered by pulse infusion, particularly with declining doses of theantibody. The dosing can be given by injections, such as intravenous orsubcutaneous injections, which may depend in part on whether theadministration is brief or chronic.

For the prevention or treatment of a disease, the appropriate dosage ofan antibody may depend on the type of disease to be treated, theseverity and the course of the disease, whether the antibody isadministered for preventive or therapeutic purposes, previous therapy,the patient's clinical history and response to the antibody, and thediscretion of the attending physician.

Depending on the type and severity of disease, about 1 μg/kg to 150mg/kg (e.g., 0.1-20 mg/kg) of antibody can be an initial candidatedosage for administration to a patient, whether, for example, by one ormore separate administrations, or by continuous infusion. A typicaldaily dosage may range from about 1 μg/kg to 100 mg/kg or more,depending on such factors as those mentioned above. An antibody-drugconjugate can be administered from about 1 μg/kg to 50 mg/kg, typicallyfrom about 0.1-20 mg/kg, whether, for example, by one or more separateadministrations, or by continuous infusion. A typical daily dosage mayrange from about 0.1 mg/kg to 10 mg/kg, or from about 0.3 mg/kg to about7.5 mg/kg, depending on such factors as those mentioned above. Forrepeated administrations over several days or longer, depending on thecondition, the treatment can be sustained until a desired suppression ofdisease symptoms occurs. However, other dosage regimens may be useful.Therapy progress can be monitored by conventional techniques and assays.

Antibody composition can be formulated, dosed, and administered in amanner consistent with good medical practice. Factors for considerationin this context include the particular disorder being treated, theparticular mammal being treated, the clinical condition of theindividual patient, the cause of the disorder, the site of delivery ofthe agent, the method of administration, the scheduling ofadministration, and other factors known to medical practitioners.

An antibody may optionally be formulated with, or administered with, oneor more therapeutic agents used to prevent or treat the disorder inquestion. For example, an antibody can be administered as a co-therapywith a standard of care therapeutic for the specific disease beingtreated.

b. Other Immunotherapy

An “immunogenic peptide” is a peptide that comprises an allele-specificmotif such that the peptide typically will bind an MHC allele (HLA inhuman) and be capable of inducing a CTL (cytotoxic T-lymphocytes)response. Thus, immunogenic peptides typically are capable of binding toan appropriate class I or II MHC molecule and inducing a cytotoxic Tcell or T helper cell response against the antigen from which theimmunogenic peptide is derived.

Peptides derived from a CAT protein can be modified to increase theirimmunogenicity, such as by enhancing the binding of the peptide to theMHC molecules in which the peptide is presented. The peptide or modifiedpeptide can be conjugated to a carrier molecule to enhance theantigenicity of the peptide. Examples of carrier molecules, include, butare not limited to, human albumin, bovine albumin, lipoprotein andkeyhole limpet hemo-cyanin (“Basic and Clinical Immunology” (1991)Stites and Terr (eds) Appleton and Lange, Norwalk Conn., San Mateo,Calif.).

Further, amino acid sequence variants of a peptide can be prepared, suchas by altering the nucleic acid sequence of the DNA which encodes thepeptide, or by peptide synthesis. At the genetic level, these variantscan be prepared by, for example, site-directed mutagenesis ofnucleotides in the DNA encoding the peptide, thereby producing DNAencoding the variant, and thereafter expressing the DNA in recombinantcell culture. The variants can exhibit the same qualitative biologicalactivity as the nonvariant peptide.

Exemplary embodiments of the invention provide peptides or modifiedpeptides derived from a CAT protein that are differentially expressed indisease. Examples of peptide modifications include, but are not limitedto, substitutions, deletions, or additions of one or more amino acids ina given immunogenic peptide sequence, or mutation of existing aminoacids within a given immunogenic peptide sequence, or derivatization ofexisting amino acids within a given immunogenic peptide sequence. Anyamino acid in an immunogenic peptide sequence may be modified. In someembodiments, at least one amino acid can be substituted or replacedwithin the given immunogenic peptide sequence. Any amino acid may beused to substitute or replace a given amino acid within the immunogenicpeptide sequence. Modified peptides can include any immunogenic peptideobtained from differentially expressed proteins, which has been modifiedand exhibits enhanced binding to the MHC molecule with which itassociates when presented to a T-cell. These modified peptides can besynthetically or recombinantly produced by conventional methods, forexample.

In certain exemplary embodiments of the invention, the peptidescomprise, or consist of, sequences of about 5-30 amino acids in lengthwhich are immunogenic (i.e., capable of inducing an immune response wheninjected into a subject).

In certain exemplary embodiments, the peptides may be used, for example,to treat T cell-mediated pathologies. The term “T cell-mediatedpathologies” refers to any condition in which an inappropriate T cellresponse is a component of the pathology. The term is intended toencompass both T cell mediated diseases and diseases resulting fromunregulated clonal T cell replication.

Modified (e.g., recombinant) or natural CAT proteins, or fragmentsthereof, can be used as a vaccine either prophylactically ortherapeutically. When provided prophylactically, a vaccine can beprovided in advance of any evidence of disease. The prophylacticadministration of a disease vaccine may serve to prevent or attenuate adisease in a mammal such as a human.

An exemplary vaccine formulation can comprise an immunogen that inducesan immune response directed against a disease-associated antigen such asa CAT protein. For example, a substantially or partially purified CATprotein or fragments thereof can be administered as a vaccine in apharmaceutically acceptable carrier. An immunogen can be administered ina pure or substantially pure form, or can be administered as apharmaceutical composition, formulation, or preparation. Exemplary dosesof protein that can be administered are about 0.001 to about 100 mg perpatient, or about 0.01 to about 100 mg per patient. Immunization can berepeated as necessary until a sufficient titer of anti-immunogenantibody or immune cells has been obtained.

Vaccine can be prepared using, for example, recombinant protein orexpression vectors comprising a nucleic acid sequence encoding all orpart of a CAT protein. Examples of vectors that can be used in vaccinesinclude, but are not limited to, defective retroviral vectors,adenoviral vectors vaccinia viral vectors, fowl pox viral vectors, orother viral vectors (Mulligan, R. C., (1993) Science 260:926-932). Thevectors can be introduced into a mammal (e.g., a human) either prior toany evidence of a disease or to mediate regression of a disease in amammal afflicted with the disease. Examples of methods for administeringa viral vector into mammals include, but are not limited to, exposure ofcells to the virus ex vivo, or injection of the retrovirus or a producercell line of the virus into the affected tissue, or intravenousadministration of the virus. Alternatively, the vector can beadministered locally by direct injection into a disease lesion ortopical application in a pharmaceutically acceptable carrier. Thequantity of viral vector to be administered can be based on the titer ofvirus particles. An exemplary range can be about 10⁶ to about 10¹¹ virusparticles per mammal.

After immunization, the efficacy of the vaccine can be assessed by, forexample, the production of antibodies or immune cells that recognize theantigen, as assessed by specific lytic activity, specific cytokineproduction, or disease regression, which can be measured usingconventional methods. If the mammal to be immunized is already afflictedwith a disease, the vaccine can be administered in conjunction withother therapeutic treatments. Examples of other therapeutic treatmentsinclude, but are not limited to, adoptive T cell immunotherapy andcoadministration of cytokines or other therapeutic drugs.

In certain embodiments, mammals, preferably humans, at high risk fordisease, especially cancer, are prophylactically treated with vaccinesof the invention. Examples include, but are not limited to, individualswith a family history of a disease, individuals who themselves have ahistory of disease (e.g., cancer that has been previously resected andat risk for reoccurrence), or individuals already afflicted with adisease. When provided therapeutically, a vaccine can be provided toenhance the patient's own immune response to a disease antigen. Anexemplary vaccine, which acts as an immunogen, can be a cell, celllysate from cells transfected with a recombinant expression vector, or aculture supernatant containing the expressed protein, for example.Alternatively, an immunogen can be, for example, a partially orsubstantially purified recombinant protein, peptide, or analog thereof,or a modified protein, peptide, or analog thereof. The proteins orpeptides can be, for example, conjugated with lipoprotein oradministered in liposomal form or with adjuvant.

Vaccination can be carried out using conventional methods. For example,an immunogen can be used in a suitable diluent such as saline or water,or complete or incomplete adjuvants. Further, an immunogen may or maynot be bound to a carrier, including carriers to increase theimmunogenicity of the immunogen. Examples of carrier molecules include,but are not limited to, bovine serum albumin (BSA), keyhole limpethemocyanin (KLH), tetanus toxoid, and the like. An immunogen also may becoupled with lipoproteins or administered in liposomal form or withadjuvants. An immunogen can be administered by any route appropriate forantibody production such as intravenous, intraperitoneal, intramuscular,subcutaneous, and the like. An immunogen can be administered once or atperiodic intervals until a significant titer of anti-CAT immune cells oranti-CAT antibody is produced. The presence of anti-CAT immune cells canbe assessed by measuring the frequency of precursor CTL (cytotoxicT-lymphocytes) against CAT antigen prior to and after immunization by aCTL precursor analysis assay (Coulie et al., 1992, International JournalOf Cancer 50:289-297). An immunoassay can be used to detect antibody inserum.

The safety of a vaccine can be determined by examining the effect ofimmunization on the general health of an immunized animal (e.g., weightchange, fever, change in appetite or behavior, etc.) and looking forpathological changes during autopsies. After initial testing in animals,a vaccine can be tested in patients having a disease of interest.Conventional methods can be used to evaluate the immune response of apatient to determine the efficiency of the vaccine.

In certain exemplary embodiments of the invention, a CAT protein orfragments thereof, or a modified CAT protein, can be exposed todendritic cells cultured in vitro. The cultured dendritic cells providea means of producing T-cell dependent antigens comprised of dendriticcell-modified antigen or dendritic cells pulsed with antigen, in whichthe antigen is processed and expressed on the antigen-activateddendritic cell. The antigen-activated dendritic cells or processeddendritic cell antigens can be used as immunogens for vaccines or forthe treatment of diseases. The dendritic cells can be exposed to theantigen for sufficient time to allow the antigens to be internalized andpresented on the surface of dendritic cells. The resulting dendriticcells or the dendritic cell-processed antigens can then be administeredto an individual in need of therapy. Such methods are described inSteinman et al. (WO93/208185) and in Banchereau et al. (EPO Application0563485A1).

In certain exemplary embodiments of the invention, T-cells isolated fromindividuals can be exposed to a CAT protein or fragment thereof, or amodified CAT protein, in vitro and then administered in atherapeutically effective amount to a patient in need of such treatment.Examples of where T-lymphocytes can be isolated include, but are notlimited to, peripheral blood cells lymphocytes (PBL), lymph nodes, ortumor infiltrating lymphocytes (TIL). Such lymphocytes can be isolatedfrom the individual to be treated or from a donor by methods known inthe art and cultured in vitro (Kawakami et al., 1989, J. Immunol. 142:2453-3461). Lymphocytes can be cultured in media such as RPMI or RPMI1640 or AIM V for 1-10 weeks. Viability can be assessed by trypan bluedye exclusion assay. Examples of how these sensitized T-cells can beadministered to a mammal include, but are not limited to, intravenously,intraperitoneally, or intralesionally. Parameters that can be assessedto determine the efficacy of these sensitized T-lymphocytes include, butare not limited to, production of immune cells in the mammal beingtreated or tumor regression. Conventional methods can be used to assessthese parameters. Such treatment can be given in conjunction withcytokines or gene-modified cells, for example (Rosenberg et al., 1992,Human Gene Therapy, 3: 75-90; Rosenberg et al., 1992, Human GeneTherapy, 3: 57-73).

9. Screening Methods Using CAT Proteins

Exemplary embodiments of the invention provide methods of screening foragents (interchangeably referred to by such terms as candidate agents,compounds, or candidate compounds) that modulate CAT protein activity(interchangeably referred to as protein function). Examples of candidateagents include, but are not limited to, proteins, peptides, antibodies,nucleic acids (such as antisense and RNAi nucleic acid molecules), andsmall molecules. Exemplary embodiments of the invention further provideagents identified by these screening methods, and methods of using theseagents, such as for treating diseases, especially cancer, particularlythe cancers identified in Table 1 (as indicated for each peptide) and/orTable 3.

Exemplary screening methods can typically comprise the steps of (i)contacting a CAT protein with a candidate agent, and (ii) assaying forCAT protein activity, wherein a change in protein activity in thepresence of the agent relative to protein activity in the absence of theagent indicates that the agent modulates CAT protein activity.

Other exemplary screening methods can determine a candidate agent'sability to modulate CAT expression. Exemplary methods can typicallycomprise the steps of (i) contacting a candidate agent with a systemthat is capable of expressing CAT protein or CAT mRNA, and (ii) assayingfor the level of CAT protein or CAT mRNA, wherein a change in the levelin the presence of the agent relative to the level in the absence of theagent indicates that the agent modulates CAT expression levels.

Exemplary embodiments of the invention further provide methods to screenfor agents that bind to CAT proteins. Exemplary methods can typicallycomprise the steps of contacting a CAT protein with a test agent andmeasuring the extent of binding of the agent to the CAT protein.

CAT proteins can be used to identify agents that modulate activity of aprotein in its natural state or an altered form that causes a specificdisease or pathology. CAT proteins and appropriate variants andfragments can be used in high-throughput screens to assay candidatecompounds for their ability to bind to CAT. These compounds can befurther screened against functional CAT proteins to determine the effectof the compound on the protein's activity. Further, these compounds canbe tested in animal or invertebrate systems to determineactivity/effectiveness. Compounds can be identified that activate(agonist) or inactivate (antagonist) CAT proteins to a desired degree.

CAT proteins can be used to screen agents for their ability to stimulateor inhibit interaction between a CAT protein and a target molecule thatnormally interacts with the CAT protein (e.g., a substrate, anextracellular binding ligand, or a component of a signal pathway that aCAT protein normally interacts with such as a cytosolic signal protein).Exemplary assays can include the steps of combining a CAT protein orfragment thereof with a candidate compound under conditions that allowthe CAT protein (or fragment thereof) to interact with a targetmolecule, and detecting the formation of a complex between the CATprotein and the target molecule or detecting the biochemical consequenceof the interaction between the CAT protein and the target molecule, suchas any of the associated effects of signal transduction (e.g., proteinphosphorylation, cAMP turnover, adenylate cyclase activation, etc.). Anyof the biological or biochemical functions mediated by a CAT protein canbe used as an endpoint assay to identify an agent that modulates CATactivity.

Candidate compounds or agents include, but are not limited to, 1)peptides such as soluble peptides, including Ig-tailed fusion peptidesand members of random peptide libraries (see, e.g., Lam et al., Nature354:82-84 (1991); Houghten et al., Nature 354:84-86 (1991)) andcombinatorial chemistry-derived molecular libraries made of D- and/orL-configuration amino acids; 2) phosphopeptides (e.g., members of randomand partially degenerate, directed phosphopeptide libraries, see, e.g.,Songyang et al., Cell 72:767-778 (1993)); 3) antibodies (e.g.,polyclonal, monoclonal, humanized, anti-idiotypic, chimeric, and singlechain antibodies as well as Fab, F(ab′)₂, Fab expression libraryfragments, and epitope-binding fragments of antibodies); and 4) smallorganic and inorganic molecules (e.g., molecules obtained fromcombinatorial and natural product libraries).

An exemplary candidate compound or agent is a soluble fragment of a CATthat competes for substrate binding. Other exemplary candidate compoundsinclude mutant CAT proteins or appropriate fragments containingmutations that affect CAT function and thus compete for substrate.Accordingly, a fragment that competes for substrate, for example with ahigher affinity, or a fragment that binds substrate but does not allowrelease, is encompassed by the invention.

Compounds can also be screened by using chimeric proteins in which anyportion of a protein such as an amino terminal extracellular domain, atransmembrane domain (e.g., transmembrane segments or intracellular orextracellular loops), or a carboxy terminal intracellular domain can bereplaced in whole or part by heterologous domains or subregions. Forexample, a substrate-binding region can be used that interacts with adifferent substrate than the substrate that is recognized by a nativetarget protein. Accordingly, a different set of signal transductioncomponents can be available as an end-point assay for activation,thereby allowing assays to be performed in other than the specific hostcell from which a target is derived.

Competition binding assays can also be used to screen for compounds thatinteract with a target protein (e.g., binding partners and/or ligands).For example, a test compound can be exposed to a target protein underconditions that allow the test compound to bind or otherwise interactwith the target protein. Soluble target protein can also be added to themixture. If the test compound interacts with the soluble target protein,it can decrease the amount of complex formed or activity of the targetprotein. This type of assay is particularly useful in instances in whichcompounds are sought that interact with specific regions of a targetprotein. Thus, the soluble target protein that competes with the targetprotein can contain peptide sequences corresponding to the target regionof interest.

To perform cell-free drug screening assays, it may be desirable toimmobilize either a CAT protein (or fragment thereof) or a molecule thatbinds the CAT protein (referred to herein as a “binding partner”) tofacilitate separation of complexes from uncomplexed forms, as well as tofacilitate automation of the assays.

Techniques for immobilizing proteins on matrices can be utilized inexemplary drug screening assays. In exemplary embodiments, a fusionprotein can be provided which adds a domain that allows a protein to bebound to a matrix. For example, glutathione-S-transferase fusionproteins can be adsorbed onto glutathione SEPHAROSE beads (SigmaChemical, St. Louis, Mo.) or glutathione derivatized microtitre plates,which are then combined with cell lysates (e.g., ³⁵S-labeled) and acandidate compound, and the mixture incubated under conditions conduciveto complex formation (e.g., at physiological conditions for salt andpH). Following incubation, the beads can be washed to remove any unboundlabel, and the matrix immobilized and radiolabel determined directly, orin the supernatant after the complexes are dissociated. Alternatively,the complexes can be dissociated from the matrix, separated by SDS-PAGE,and the level of a binding partner found in the bead fractionquantitated from the gel using standard electrophoretic techniques. Forexample, either a target protein or a binding partner can be immobilizedby conjugation of biotin and streptavidin using techniques well known inthe art. Alternatively, antibodies that are reactive with a targetprotein but do not interfere with binding of the target protein to itsbinding partner can be derivatized to the wells of a plate, and thetarget protein trapped in the wells by antibody conjugation.Preparations of a binding partner and a candidate compound can beincubated in target protein-presenting wells and the amount of complextrapped in the well can be quantitated. Methods for detecting suchcomplexes, in addition to those described for GST-immobilized complexes,include immunodetection of complexes using antibodies reactive with abinding partner, or which are reactive with a target protein and competewith the binding partner, as well as target protein-linked assays whichrely on detecting an enzymatic activity associated with a bindingpartner.

In exemplary embodiments of the invention, a CAT protein can be used asa “bait protein” in a two-hybrid assay or three-hybrid assay (see, e.g.,U.S. Pat. No. 5,283,317; Zervos et al. (1993) Cell 72:223-232; Madura etal. (1993) J. Biol. Chem. 268:12046-12054; Bartel et al. (1993)Biotechniques 14:920-924; Iwabuchi et al. (1993) Oncogene 8:1693-1696;and Brent WO94/10300), to identify other proteins, which bind to orinteract with a CAT protein and are involved in the protein's activity.The two-hybrid system is based on the modular nature of mosttranscription factors, which typically consist of separable DNA-bindingand activation domains. In exemplary embodiments, the two-hybrid assaycan utilize two different DNA constructs. In one construct, a gene thatencodes a CAT protein can be fused to a gene encoding the DNA bindingdomain of a known transcription factor (e.g., GAL-4). In the otherconstruct, a DNA sequence from a library of DNA sequences that encode anunidentified protein (“prey” or “sample”) can be fused to a gene thatencodes the activation domain of the known transcription factor. If the“bait” and the “prey” proteins are able to interact in vivo, forming aCAT-dependent complex, the DNA-binding and activation domains of thetranscription factor are brought into close proximity. This proximityallows transcription of a reporter gene (e.g., LacZ), which can beoperably linked to a transcriptional regulatory site responsive to thetranscription factor. Expression of the reporter gene can be detectedand cell colonies containing the functional transcription factor can beisolated and used to obtain the cloned gene that encodes the proteinthat interacts with the CAT protein.

Agents that modulate a CAT protein can be identified using one or moreof the above assays, alone or in combination. For example, a cell-basedor cell free system can be used for initial identification of agents,and then activity of the agents can be confirmed in an animal or othermodel system. Such model systems are well known in the art and canreadily be employed in this context.

10. Diagnosis, Treatment, and Screening Methods Using CAT Nucleic AcidMolecules

The nucleic acid molecules of the invention are useful, for example, asprobes, primers, chemical intermediates, and in biological assays. Thenucleic acid molecules are useful as hybridization probes for messengerRNA, transcript/cDNA, and genomic DNA to detect or isolate full-lengthcDNA and genomic clones encoding a CAT protein, or variants thereof. Thenucleic acid molecules are also useful as primers for PCR to amplify anygiven region of a nucleic acid molecule and are useful to synthesizeantisense molecules of desired length and sequence. The nucleic acidmolecules are also useful for producing ribozymes corresponding to all,or a part, of the mRNA produced from the nucleic acid moleculesdescribed herein.

The nucleic acid molecules are also useful for constructing recombinantvectors. Exemplary vectors include expression vectors that express aportion of, or all of, a CAT protein. The nucleic acid molecules arealso useful for expressing antigenic portions of the proteins. Thenucleic acid molecules are also useful for constructing host cellsexpressing a part, or all, of the proteins. The nucleic acid moleculesare also useful for constructing transgenic animals expressing all, or apart, of the proteins.

A primer or probe can correspond to any sequence along the entire lengthof a CAT-encoding nucleic acid molecule such as the nucleic acidmolecules of SEQ ID NOS:723-1281, 1976, and 1978. Accordingly, a primeror probe can be derived from 5′ noncoding regions, coding regions, or 3′noncoding regions, for example.

Exemplary in vitro techniques for detection of mRNA include Northernhybridizations and in situ hybridizations. Exemplary in vitro techniquesfor detecting DNA include Southern hybridizations and in situhybridization. Reverse transcriptase PCR amplification (RT-PCR) and thelike can also be used for detecting RNA expression. A specific exemplarymethod of detection comprises using TaqMan technology (AppliedBiosystems, Foster City, Calif.).

a. Methods of Diagnosis Using Nucleic Acids

Nucleic acid molecules of the invention are useful, for example, ashybridization probes for determining the presence, level, form, and/ordistribution of nucleic acid expression. Exemplary probes can be used todetect the presence of, or to determine levels of, a specific nucleicacid molecule in cells, tissues, and in organisms. Accordingly, probescorresponding to a CAT described herein can be used to assess expressionand/or gene copy number in a given cell, tissue, or organism, which canbe applied to, for example, diagnosis of disorders involving an increaseor decrease in CAT protein expression relative to normal CAT proteinexpression levels.

Probes can be used as part of a diagnostic test kit for identifyingcells or tissues that express CAT protein differentially, such as bymeasuring a level of a CAT-encoding nucleic acid (e.g., mRNA or genomicDNA) in a sample of cells from a subject, or determining if aCAT-encoding nucleic acid is mutated.

Exemplary embodiments of the invention encompass kits for detecting thepresence of CAT-encoding nucleic acid (e.g., mRNA or genomic DNA) in abiological sample. For example, an exemplary kit can comprise reagentssuch as a labeled or labelable nucleic acid or agent capable ofdetecting CAT nucleic acid in a biological sample; means for determiningthe amount of CAT nucleic acid in the sample; and means for comparingthe amount of CAT nucleic acid in the sample with a standard. Thecompound or agent can be packaged in a suitable container. The kit canfurther comprise instructions for using the kit to detect CAT nucleicacid.

The nucleic acid molecules are useful in diagnostic assays forqualitative changes in CAT nucleic acid expression, and particularly inqualitative changes that lead to pathology. The nucleic acid moleculescan be used to detect mutations in CAT genes and gene expressionproducts such as mRNA. The nucleic acid molecules can be used ashybridization probes to detect naturally occurring genetic mutations ina CAT gene and to determine whether a subject with the mutation is atrisk for a disorder caused by the mutation. Examples of mutationsinclude deletions, additions, or substitutions of one or morenucleotides in a gene, chromosomal rearrangements (such as inversions ortranspositions), and modification of genomic DNA such as aberrantmethylation patterns or changes in gene copy number (such asamplification). Detection of a mutated form of a CAT gene associatedwith a dysfunction can provide a diagnostic tool for an active diseaseor susceptibility to disease in instances in which the disease resultsfrom overexpression, underexpression, or altered expression of a CATprotein, for example.

Mutations in a CAT gene can be detected at the nucleic acid level by avariety of techniques. For example, genomic DNA, RNA, or cDNA can beanalyzed directly or can be amplified (e.g., using PCR) prior toanalysis. In certain exemplary embodiments, detection of a mutationinvolves the use of a probe/primer in a PCR reaction (see, e.g. U.S.Pat. Nos. 4,683,195 and 4,683,202), such as anchor PCR or RACE PCR, or,alternatively, in a ligation chain reaction (LCR) (see, e.g., Landegranet al., Science 241:1077-1080 (1988) and Nakazawa et al., PNAS91:360-364 (1994)), the latter of which can be particularly useful fordetecting point mutations in a gene (see Abravaya et al., Nucleic AcidsRes. 23:675-682 (1995)). Exemplary methods such as these can include thesteps of collecting a sample of cells from a patient, isolating nucleicacid (e.g., genomic, mRNA, or both) from the cells of the sample,contacting the nucleic acid with one or more primers which specificallyhybridize to a target nucleic acid under conditions such thathybridization and amplification of the target nucleic acid (if present)occurs, and detecting the presence or absence of an amplificationproduct, or detecting the size of the amplification product andcomparing the length to a control sample. Deletions and insertions canbe detected by a change in size of the amplified product compared to anormal genotype. Point mutations can be identified by hybridizingamplified DNA to normal RNA or antisense DNA sequences, for example.

Alternatively, mutations in a CAT gene can be identified, for example,by alterations in restriction enzyme digestion patterns as determined bygel electrophoresis. Further, sequence-specific ribozymes (U.S. Pat. No.5,498,531) can be used to identify the presence of specific mutations bydevelopment or loss of a ribozyme cleavage site. Perfectly matchedsequences can be distinguished from mismatched sequences by nucleasecleavage digestion assays or by differences in melting temperature.

Sequence changes at specific locations can be assessed by nucleaseprotection assays such as RNase and S1 protection, or chemical cleavagemethods. Furthermore, sequence differences between a mutant CAT gene anda corresponding wild-type gene can be determined by direct DNAsequencing. A variety of automated sequencing procedures can be utilizedwhen performing diagnostic assays (Naeve, C. W., (1995) Biotechniques19:448), including sequencing by mass spectrometry (e.g., PCTInternational Publication No. WO 94/16101; Cohen et al., Adv.Chromatogr. 36:127-162 (1996); and Griffin et al., Appl. Biochem.Biotechnol. 38:147-159 (1993)).

Other methods for detecting mutations in a nucleic acid include methodsin which protection from cleavage agents is used to detect mismatchedbases in RNA/RNA or RNA/DNA duplexes (Myers et al., Science 230:1242(1985)); Cotton et al., PNAS 85:4397 (1988); Saleeba et al., Meth.Enzymol. 217:286-295 (1992)), electrophoretic mobility of mutant andwild type nucleic acid is compared (Orita et al., PNAS 86:2766 (1989);Cotton et al., Mutat. Res. 285:125-144 (1993); and Hayashi et al.,Genet. Anal. Tech. Appl. 9:73-79 (1992)), and movement of mutant orwild-type fragments in polyacrylamide gels containing a gradient ofdenaturant is assayed using denaturing gradient gel electrophoresis(DGGE) (Myers et al., Nature 313:495 (1985)). Examples of othertechniques for detecting point mutations include selectiveoligonucleotide hybridization, selective amplification, and selectiveprimer extension.

b. Methods of Monitoring Treatment and Pharmacogenomic Methods UsingNucleic Acids

Nucleic acid molecules of the invention are also useful for monitoringthe effectiveness of modulating agents on the expression or activity ofa CAT gene, such as in clinical trials or in a treatment regimen. Forexample, the gene expression pattern of a CAT gene can serve as abarometer for the continuing effectiveness of treatment with a compound,particularly with compounds to which a patient can develop resistance.The gene expression pattern can also serve as a marker indicative of aphysiological response of the affected cells to the compound. Forexample, based on monitoring nucleic acid expression, the administrationof a compound can be increased or alternative compounds to which thepatient has not become resistant can be administered instead. Similarly,if the level of nucleic acid expression falls below a desirable level,administration of the compound can be commensurately decreased.

The nucleic acid molecules are also useful for testing an individual fora genotype that, while not necessarily causing a disease, neverthelessaffects the treatment modality. Thus, the nucleic acid molecules can beused to study the relationship between an individual's genotype and theindividual's response to a compound used for treatment (pharmacogenomicrelationship). Accordingly, the nucleic acid molecules provided hereincan be used to assess the mutation content of a target gene in anindividual in order to select an appropriate compound or dosage regimenfor treatment. For example, target nucleic acid molecules having geneticvariations that affect treatment can provide diagnostic targets that canbe used to tailor treatment to an individual. Accordingly, theproduction of recombinant cells and animals having these geneticvariations allows effective clinical design of treatment compounds anddosage regimens, for example.

c. Methods of Treatment Using Nucleic Acids

Nucleic acid molecules of the invention are useful to design antisenseconstructs to control CAT gene expression in cells, tissues, andorganisms. An antisense nucleic acid molecule typically blockstranslation of mRNA into CAT protein by hybridizing to target mRNA in asequence-specific manner. Nucleic acid molecules of the invention canalso be used to specifically suppress gene expression by methods such asRNA interference (RNAi). RNAi and antisense-based gene suppression arewell known in the art (e.g., Science 288:1370-1372, 2000). RNAitypically operates on a post-transcriptional level and is sequencespecific. RNAi and antisense nucleic acid molecules are useful fortreating diseases, especially cancer. RNAi fragments, particularlydouble-stranded (ds) RNAi, as well as antisense nucleic acid moleculescan also be used to generate loss-of-function phenotypes by suppressinggene expression. Accordingly, exemplary embodiments of the inventionprovide RNAi and antisense nucleic acid molecules, and methods of usingthese RNAi and antisense nucleic acid molecules, such as for therapy orfor modulating cell function. Nucleic acid molecules may also beproduced that are complementary to a region of a gene involved intranscription, such as to hybridize to the gene to preventtranscription.

Exemplary embodiments of the invention relate to isolated RNA molecules(double-stranded; single-stranded) that are about 17 to about 29nucleotides (nt) in length, and more particularly about 21 to about 25nt in length, which mediate RNAi (e.g., degradation of mRNA, and suchmRNA may be referred to herein as mRNA to be degraded). With respect toRNAi, the terms RNA, RNA molecule(s), RNA segment(s), and RNAfragment(s) are used interchangeably to refer to RNA that mediates RNAi.These terms include double-stranded RNA, single-stranded RNA, isolatedRNA (e.g., partially purified RNA, essentially pure RNA, synthetic RNA,recombinantly produced RNA), as well as altered RNA that differs fromnaturally occurring RNA by the addition, deletion, substitution, and/oralteration of one or more nucleotides. Such alterations can include, forexample, addition of non-nucleotide material, such as to the end(s) of a21-25 nt RNA or internally (at one or more nucleotides of the RNA).Nucleotides in exemplary RNA molecules of the invention can alsocomprise non-standard nucleotides, including non-naturally occurringnucleotides or deoxyribonucleotides. Collectively, all such altered RNAsare referred to as analogs or analogs of naturally-occurring RNA. RNA of21-25 nt typically need only be sufficiently similar to natural RNA thatit has the ability to mediate RNAi. As used herein, the phrase “mediatesRNAi” refers to the ability to distinguish which RNAs are to be degradedby RNAi processes. RNA that mediates RNAi directs degradation ofparticular mRNAs by RNAi processes. Such RNA may include RNAs of variousstructures, including short hairpin RNA.

In certain exemplary embodiments, the invention relates to RNA moleculesof about 21 to about 25 nt that direct cleavage of specific mRNA towhich their sequence corresponds. It is not necessary that there be aperfect correspondence (i.e., match) of the sequences, but thecorrespondence must be sufficient to enable the RNA to direct RNAicleavage of the target mRNA (Holen et al., Nucleic Acids Res.33:4704-4710 (2005)). In an exemplary embodiment, the 21-25 nt RNAmolecules of the invention comprise a 3′ hydroxyl group.

Certain exemplary embodiments of the invention relate to 21-25 nt RNAsof specific genes, produced by chemical synthesis or recombinant DNAtechniques, that mediate RNAi. As used herein, the term “isolated RNA”includes RNA obtained by any means, including processing or cleavage ofdsRNA, production by chemical synthetic methods, and production byrecombinant DNA techniques, for example. Exemplary embodiments of theinvention further relate to uses of the 21-25 nt RNAs, such as fortherapeutic or prophylactic treatment and compositions comprising 21-25nt RNAs that mediate RNAi, such as pharmaceutical compositionscomprising 21-25 nt RNAs and an appropriate carrier.

Further exemplary embodiments of the invention relate to methods ofmediating RNAi of genes of a patient. For example, RNA of about 21 toabout 25 nt which targets a specific mRNA to be degraded can beintroduced into a patient's cells. The cells can be maintained underconditions allowing degradation of the mRNA, resulting in RNA-mediatedinterference of the mRNA of the gene in the cells of the patient.Treatment of cancer patients, for example, with RNAi may inhibit thegrowth and spread of the cancer and reduce tumor size. Treatment ofpatients using RNAi can also be in combination with other therapies. Forexample, RNAi can be used in combination with other treatmentmodalities, such as chemotherapy, radiation therapy, and othertreatments. In an exemplary embodiment, a chemotherapy agent is used incombination with RNAi. In a further exemplary embodiment, GEMZAR(gemcitabine HCl) chemotherapy is used with RNAi.

Treatment of certain diseases by RNAi may require introduction of theRNA into the disease cells. RNA can be directly introduced into a cell,or introduced extracellularly into a cavity, interstitial space, intothe circulation of a patient, or introduced orally, for example.Physical methods of introducing nucleic acids, such as injectiondirectly into a cell or extracellular injection into a patient, may alsobe used. RNA may be introduced into vascular or extravascularcirculation, the blood or lymph system, or the cerebrospinal fluid, forexample. RNA may be introduced into an embryonic stem cell or anothermultipotent cell, which may be derived from a patient. Physical methodsof introducing nucleic acids include injection of a solution containingthe RNA, bombardment by particles covered by the RNA, soaking cells ortissue in a solution of the RNA, or electroporation of cell membranes inthe presence of the RNA. A viral construct packaged into a viralparticle may be used to introduce an expression construct into a cell,with the construct expressing the RNA. Other methods known in the artfor introducing nucleic acids to cells may be used, such aslipid-mediated carrier transport, chemical-mediated transport, and thelike. The RNA may be introduced along with components that perform oneor more of the following activities: enhance RNA uptake by the cell,promote annealing of the duplex strands, stabilize the annealed strands,or otherwise increase inhibition of the target gene.

Exemplary RNA of the invention can be used alone or as a component of akit having at least one reagent for carrying out in vitro or in vivointroduction of the RNA to a cell, tissue/fluid, or patient. Exemplarycomponents of a kit include dsRNA and a vehicle that promotesintroduction of the dsRNA. A kit may also include instructions for usingthe kit.

Certain exemplary embodiments of the invention provide compositions andmethods for cleavage of mRNA by ribozymes having nucleotide sequencescomplementary to one or more regions in the mRNA, thereby attenuatingthe translation of the mRNA. Examples of regions in mRNA that can betargeted by ribozymes include coding regions, particularly codingregions corresponding to catalytic or other functional activities of atarget protein, such as substrate binding. These compositions andmethods may be used to treat a disorder characterized by abnormal orundesired target nucleic acid expression.

In certain exemplary embodiments, nucleic acid molecules of theinvention may be used for gene therapy in individuals having cells thatare aberrant in gene expression of a target. For example, recombinantcells that have been engineered ex vivo (which can include anindividual's own cells) can be introduced into an individual where thecells produce the desired target protein to thereby treat theindividual.

d. Methods of Screening Using Nucleic Acids

Nucleic acid expression assays are useful for drug screening to identifycompounds that modulate CAT nucleic acid expression.

Exemplary embodiments of the invention thus provide methods foridentifying a compound that can be used to treat a disease associatedwith differential expression of a CAT gene, especially cancer. Exemplarymethods can typically include assaying the ability of a compound tomodulate the expression of a target nucleic acid to thereby identify acompound that can be used to treat a disorder characterized by undesiredtarget nucleic acid expression. The assays can be performed incell-based or cell-free systems. Examples of cell-based assays includecells naturally expressing target nucleic acid or recombinant cellsgenetically engineered to express specific target nucleic acidsequences.

Assays for target nucleic acid expression can involve direct assay oftarget nucleic acid levels, such as mRNA levels, or on collateralcompounds involved in a signal pathway. Further, the expression of genesthat are up- or down-regulated in response to a signal pathway can alsobe assayed. In these embodiments, the regulatory regions of these genescan be operably linked to a reporter gene such as luciferase.

Thus, in exemplary embodiments, modulators of gene expression of atarget can be identified in methods wherein a cell is contacted with acandidate agent and the expression of target mRNA determined. The levelof expression of target mRNA in the presence of the candidate agent iscompared to the level of expression of target mRNA in the absence of thecandidate agent. The candidate agent can then be identified as amodulator of target nucleic acid expression based on this comparison andmay be used, for example, to treat a disorder characterized by aberranttarget nucleic acid expression. When expression of target mRNA isstatistically significantly greater in the presence of the candidateagent than in its absence, the candidate agent is identified as astimulator (agonist) of nucleic acid expression. When nucleic acidexpression is statistically significantly less in the presence of thecandidate agent than in its absence, the candidate compound isidentified as an inhibitor (antagonist) of nucleic acid expression.

11. Arrays and Expression Analysis

“Array” (interchangeably referred to as “microarray”) typically refersto an arrangement of at least one, but more typically at least two,nucleic acid molecules, proteins, or antibodies on a substrate. Incertain exemplary arrangements, at least one of the nucleic acidmolecules, proteins, or antibodies typically represents a control orstandard, and other nucleic acid molecules, proteins, or antibodies areof diagnostic or therapeutic interest. In exemplary embodiments, thearrangement of nucleic acid molecules, proteins, or antibodies on thesubstrate is such that the size and signal intensity of each labeledcomplex (e.g., formed between each nucleic acid molecule and acomplementary nucleic acid, or between each protein and a ligand orantibody, or between each antibody and a protein to which the antibodyspecifically binds) is individually distinguishable.

An “expression profile” is a representation of target expression in asample. A nucleic acid expression profile can be produced using, forexample, arrays, sequencing, hybridization, or amplificationtechnologies for nucleic acids from a sample. A protein expressionprofile can be produced using, for example, arrays, gel electrophoresis,mass spectrometry, or antibodies (and, optionally, labeling moieties)which specifically bind proteins. Nucleic acids, proteins, or antibodiescan be attached to a substrate or provided in solution, and theirdetection can be based on methods well known in the art.

A substrate includes, but is not limited to, glass, paper, nylon orother type of membrane, filter, chip, metal, or any other suitable solidor semi-solid (e.g., gel) support.

Exemplary arrays can be prepared and used according to the methodsdescribed in U.S. Pat. No. 5,837,832; PCT application WO95/11995;Lockhart et al., 1996, Nat. Biotech. 14: 1675-1680; Schena et al., 1996;Proc. Natl. Acad. Sci. 93: 10614-10619; and U.S. Pat. No. 5,807,522.Exemplary embodiments of the invention also provide antibody arrays(see, e.g., de Wildt et al. (2000) Nat. Biotechnol. 18:989-94).

Certain exemplary embodiments of the invention provide a nucleic acidarray for assaying target expression, which can be composed ofsingle-stranded nucleic acid molecules, usually either syntheticantisense oligonucleotides or fragments of cDNAs, fixed to a solidsupport. The oligonucleotides can be, for example, about 6-60nucleotides in length, about 15-30 nucleotides in length, or about 20-25nucleotides in length.

To produce oligonucleotides to a target nucleic acid molecule for anarray, the target nucleic acid molecule of interest is typicallyexamined using a computer algorithm to identify oligonucleotides ofdefined length that are unique to the nucleic acid molecule, have a GCcontent within a range suitable for hybridization, and lack predictedsecondary structure that may interfere with hybridization. In certaininstances, it may be desirable to use pairs of oligonucleotides on anarray. In exemplary embodiments, the “pairs” can be identical, exceptfor one nucleotide (which can be located in the center of the sequence,for example). The second oligonucleotide in the pair (mismatched by one)serves as a control. Any number of oligonucleotide pairs may beutilized.

Oligonucleotides can be synthesized on the surface of a substrate, suchas by using a light-directed chemical process or by using a chemicalcoupling procedure and an ink jet application apparatus (e.g., PCTapplication WO95/251116).

In some exemplary embodiments, an array can be used to diagnose ormonitor the progression of disease, for example, by assaying targetexpression.

For example, an oligonucleotide probe specific for a target can belabeled by standard methods and added to a biological sample from apatient under conditions that allow for the formation of hybridizationcomplexes. After an incubation period, the sample can be washed and theamount of label (or signal) associated with hybridization complexes canbe quantified and compared with a standard value. If complex formationin the patient sample is significantly altered (higher or lower) incomparison to a normal (e.g., healthy) standard, or is similar to adisease standard, this differential expression can be diagnostic of adisorder.

By analyzing changes in patterns of target expression, disease may bediagnosed at earlier stages before a patient is symptomatic. Inexemplary embodiments of the invention, arrays or target expressionanalysis methods can be used to formulate a diagnosis or prognosis, todesign a treatment regimen, and/or to monitor the efficacy of treatment.For example, a treatment dosage can be established that causes a changein target expression patterns indicative of successful treatment, andtarget expression patterns associated with the onset of undesirable sideeffects can be avoided. In further exemplary embodiments, assays oftarget expression can be repeated on a regular basis to determine if thelevel of target expression in a patient begins to approximate that whichis observed in a normal subject. The results obtained from successiveassays may be used to show the efficacy of treatment over a periodranging from several days to years, for example.

Exemplary arrays of the invention can also be used to screen candidateagents, such as to identify agents that produce a target expressionprofile similar to that caused by known therapeutic agents, with theexpectation that agents that cause a similar expression profile of atarget may have similar therapeutic effects and/or modes of action onthe target.

EXAMPLES

Exemplary embodiments of the invention are further described in thefollowing examples, which do not limit the scope of the invention.

1. Tissue Samples and Cell Lines

Tissue Processing and Preparation of Single Cell Suspensions from Tissue

Tissue samples (e.g., normal tissues or disease tissues such assurgically resected neoplastic or metastatic lesions) can be procuredfrom clinical sites and transported in transport buffer. Tissues can becollected as remnant tissues following surgical resection of cancer (orother disease) tissues. Remnant tissues are supplied followingprocessing for pathological diagnosis according to proper standards ofpatient care. Normal tissue specimens can be normal tissue adjacent totumors (or other disease tissue) that is collected during tumorresection. Normal tissue from healthy patients not having cancer (orother disease of interest) can also be included, such as to reduce thecontribution from pre-neoplastic changes that may exist in normaladjacent tissue. Procurement of tissue samples is carried out in ananonymous manner in compliance with federally mandated ethical and legalguidelines (HIPAA) and in accordance with clinical institution ethicalreview board and internal institutional review board guidelines.

Tissue can be crudely minced and incubated for 20-30 minutes withperiodic agitation at 37° C. in Enzyme Combination #1 (200 unitscollagenase, cat #C5894 Sigma; 126 μg DNAse I, cat #D4513 Sigma (in 10mM Tris/HCl pH7.5); 50 mM NaCl; 10 mM MgCl₂; 0.05% elastase, cat #E7885Sigma) (additionally, hyaluronidase enzyme may also be utilized). D-PBSis added at 3× the volume of the enzyme combination, the tissue finelyminced, and disassociated cells passed through a 200 μm filter. Thecells are washed twice with D-PBS. Red blood cells are lysed withPharMLyse (BD Biosciences) when necessary. Cell number and viability aredetermined by PI exclusion (GUAVA). Cells at a total cell number greaterthan 20×10⁶ are sorted using a high-speed sorter (MoFlo Cytomation) forepithelial cells (EpCAM positive).

The remaining undigested tissue is incubated for 20-30 minutes withperiodic agitation at 37° C. in Enzyme Combination #2 (1× LiberaseBlendzyme 1, cat #988-417 Roche; 1× Liberase Blendzyme 3, cat #814-184Roche; 0.05% elastase, cat #E7885 Sigma). D-PBS is added at 3× thevolume of the enzyme combination, and the tissue finely minced untiltissue is completely disassociated. The cells are passed through a 200μm filter, washed twice with D-PBS, and pooled with cells from theEnzyme Combination #1 digestion.

Cells are passed through a 70 μm filter for single cell suspension, andcell number and viability are determined by PI exclusion (GUAVA). Whenneeded, red blood cells are lysed with PharMLyse (BD Biosciences). Cellsare incubated in 20 ml of 1× PharMLyse in D-PBS for 30 seconds withgentle agitation and cells pelleted at 300× g for 5 minutes at 4° C.Cells are washed once in D-PBS and cell number and viability arerecalculated by PI exclusion using the GUAVA. Cells at a total cellnumber greater than 20×10⁶ are sorted using a high-speed sorter (MoFloCytomation) for epithelial cells (EpCAM positive).

Single cell suspensions can also be prepared from tissue samples asfollows: specimens are washed in DTT for 15 min, digested with Dispase(30-60 min), then filtered twice (380 μm/74 μm) before red blood cellsare removed through addition of ACK lysis buffer. Epithelial (EpCAM) andleukocyte (CD45) content and cellular viability (PI exclusion) can bedetermined through flow cytometry analysis (LSR I, BD Biosciences, SanJose, Calif.).

The epithelial content of both disease and normal specimens can beenriched through depletion of immune CD45-positive cells by flowcytometry or purification of Epithelial Cell Surface Antigen(ECSA/EpCam)-positive cells by bead capture.

Bead capture of epithelial cells can be performed using a DynalCELLection Epithelial Enrich kit (Invitrogen, Carlsbad, Calif.) asfollows. Dynal CELLection beads at a concentration of 2×10⁸ beads areincubated with 1×10⁸ cells in HBSS with 10% fetal calf serum for 30minutes at 4° C. Cells and beads are placed in a magnet system Dynal MPCfor 2 minutes. Bead/cell complexes are washed in RPMI 1640 media with 1%fetal calf serum. Cells are released from the bead complex with 15minute incubation with DNase with agitation in RPMI with 1% fetal calfserum.

DynalBead cell depletion of CD45 cells can be carried out as follows.DynalBead M-450 CD45 beads and cells are incubated at a concentration of250 μl beads per 2×10⁷ cells for 30 minutes at 4° C. Bead/cell complexesare washed in DPBS buffer with 2% fetal bovine serum. Cells and beadsare placed in a magnet system Dynal MPC for 2 minutes. The supernatantcontains EpCAM enriched cells.

Cell Line Culture

Cell lines can be obtained from the American Type Culture Collection(ATCC, Manassas, Va.). Cell lines can be grown in a culturing mediumthat is supplemented as necessary with growth factors and serum, inaccordance with the ATCC guidelines for each particular cell line.Cultures are established from frozen stocks in which the cells aresuspended in a freezing medium (cell culture medium with 10% DMSO [v/v])and flash frozen in liquid nitrogen. Frozen stocks prepared in this wayare stored in liquid nitrogen vapor. Cell cultures are established byrapidly thawing frozen stocks at 37° C. Thawed stock cultures are slowlytransferred to a culture vessel containing a large volume ofsupplemented culture medium. For maintenance of culture, cells areseeded at 1×10⁵ cells/per ml in medium and incubated at 37° C. untilconfluence of cells in the culture vessel exceeds 50% by area. At thistime, cells are harvested from the culture vessel using enzymes or EDTAwhere necessary. The density of harvested, viable cells is estimated byhemocytometry and the culture reseeded as above. A passage of thisnature is repeated no more than 25 times, at which point the culture isdestroyed and reestablished from frozen stocks as described above.

Alternatively, cells (e.g., adipocytes such as differentiatedsubcutaneous or visceral adipocytes) can be obtained from commercialsources, which may provide the cells seeded into T-75 tissue cultureflasks. Upon arrival in the laboratory, the media is removed andreplaced with DMEM/Ham's F-12 medium (1:1 v/v) supplemented with HEPESpH 7.4, FBS, biotin, pantothenate, human insulin, dexamethasone,penicillin-streptomycin, and Amphotercin B. The cells are cultured fortwo days and then harvested with versene before enrichment of proteins.

Alternatively, for secreted protein analysis, cells can be grown underroutine tissue culture conditions in 490 cm² roller bottles at aninitial seeding density of approximately 15 million cells per rollerbottle. When the cells reach ˜70-80% confluence, the culturing media isremoved, the cells are washed 3 times with D-PBS and once with CD293protein-free media (Invitrogen cat #11913-019), and the culturing mediais replaced with CD293 for generating conditioned media. Cells areincubated for 72 hours in CD293 and the media is collected for analysis,such as mass spectrometry analysis of secreted proteins (30-300 ml).Cell debris is removed from the conditioned media by centrifugation at300 g for 5 minutes and filtering through a 0.2 micron filter prior toanalysis.

Alternatively, for secreted protein analysis, conditioned mediacollected from differentiated cells (e.g., visceral or subcutaneousadipocytes), can be obtained (e.g., from a commercial source).Conditioned medium is shipped on dry ice and maintained at −80° C. aheadof protein capture. Cells are isolated from tissue and expanded topassage 2 (P2) to passage 4 (P4) prior to differentiation. Media ischanged and cells are grown in conditioned medium for three days priorto harvesting. Enriching for proteins such as secreted proteins can thenbe carried out.

2. Cloning and Expression of Target Proteins

cDNA Retrieval

Peptide sequences can be searched using the BLAST algorithm againstrelevant protein sequence databases to identify the correspondingfull-length protein (reference sequence). Each full-length proteinsequence can then be searched using the BLAST algorithm against a humancDNA clone collection. For each sequence of interest, clones can bepulled and streaked onto LB/Ampicillin (100 μg/ml) plates. Plasmid DNAis isolated using Qiagen spin mini-prep kit and verified by restrictiondigest. Subsequently, the isolated plasmid DNA is sequence verifiedagainst the reference full-length protein sequence. Sequencing reactionsare carried out using Applied Biosystems BigDye Terminator kit followedby ethanol precipitation. Sequence data is collected using the AppliedBiosystems 3700 Genetic Analyzer and analyzed by alignment to thereference full-length protein sequence using the Clone Manager alignmenttool.

PCR

PCR primers are designed to amplify the region encoding the full-lengthprotein and/or any regions of the protein that are of interest forexpression (e.g., antigenic or hydrophilic regions as determined by theClone Manager sequence analysis tool). Primers also contain 5′ and 3′overhangs to facilitate cloning (see below). PCR reactions contain 2.5units Platinum Taq DNA Polymerase High Fidelity (Invitrogen), 50 ng cDNAplasmid template, 1 μM forward and reverse primers, 800 μM dNTP cocktail(Applied Biosystems), and 2 mM MgSO₄. After 20-30 cycles (94° C. for 30seconds, 55° C. for 1 minute, and 73° C. for 2 minutes), the resultingproduct is verified by sequence analysis and quantitated by agarose gelelectrophoresis.

Construction of Entry Clones

PCR products are cloned into an entry vector for use with the Gatewayrecombination based cloning system (Invitrogen). These vectors includepDonr221, pDonr201, pEntr/D-TOPO, or pEntr/SD/D-TOPO and are used asdescribed in the cloning methods below.

TOPO Cloning into pEntr/D-TOPO or pEntr/SD/D-TOPO

For cloning using this method, the forward PCR primer contains a 5′overhang containing the sequence “CACC”. PCR products are generated asdescribed above and cloned into the entry vector using the InvitrogenTOPO® cloning kit. Reactions are typically carried out at roomtemperature for 10 minutes and subsequently transformed into TOP10chemically competent cells (Invitrogen, CA). Candidate clones arepicked, and plasmid DNA is prepared using a Qiagen spin mini-prep kitand screened by restriction enzyme digestion. Inserts are subsequentlysequence-verified as described above.

Gateway Cloning into pDonr201 or pDonr221

For cloning using this method, PCR primers contain forward and reverse5′ overhangs. PCR products are generated as described above.Protein-encoding nucleic acid molecules are recombined into the entryvector using the Invitrogen Gateway BP Clonase enzyme mix. Reactions aretypically carried out at 25° C. for 1 hour, treated with Proteinase K at37° C. for 10 minutes, and transformed into Library Efficiency DH5αchemically competent cells (Invitrogen, CA). Candidate clones arepicked, plasmid DNA is prepared using a Qiagen spin mini-prep kit, andscreened by restriction enzyme digestion. Inserts are subsequentlysequence-verified as described above.

Construction of Expression Clones

Protein-encoding nucleic acid molecules are transferred from the entryconstruct into a series of expression vectors using the Gateway LRClonase enzyme mix. Reactions are typically carried out for 1 hour at25° C., treated with Proteinase K at 37° C. for 10 minutes, andsubsequently transformed into Library Efficiency DH5a chemicallycompetent cells (Invitrogen). Candidate clones are picked, plasmid DNAis prepared using a Qiagen spin mini-prep kit, and screened byrestriction enzyme digestion. Expression vectors include, but are notlimited to, pDest14, pDest15, pDest17, pDest8, pDest10 and pDest20.These vectors allow expression in systems such as E. coli andrecombinant baculovirus. Other vectors not listed here allow expressionin yeast, mammalian cells, or in vitro.

Expression of Recombinant Proteins in E. coli

Constructs are transformed into one or more of the following hoststrains: BL21 SI, BL21 AI, (Invitrogen), Origami B (DE3), Origami B(DE3) pLysS, Rosetta (DE3), Rosetta (DE3) pLysS, Rosetta-Gami (DE3),Rosetta-Gami (DE3) pLysS, or Rosetta-Gami B (DE3) pLysS (Novagen). Thetransformants are grown in LB with or without NaCl and with appropriateantibiotics, at temperatures in the range of 20-37° C., with aeration.Expression is induced with the addition of IPTG (0.03-0.30 mM) or NaCl(75-300 mM) when the cells are in mid-log growth. Growth is continuedfor one to 24 hours post-induction. Cells are harvested bycentrifugation in a Sorvall RC-3C centrifuge in a H6000A rotor for 10minutes at 3000 rpm at 4° C. Cell pellets are stored at −80° C.

Expression of Recombinant Proteins Using Baculovirus

Recombinant proteins are expressed using baculovirus in Sf21 fall armyworm ovarian cells. Recombinant baculoviruses are prepared using theBac-to-Bac system (Invitrogen) per the manufacturer's instructions.Proteins are expressed on the large scale in Sf900II serum-free medium(Invitrogen) in a 10 L bioreactor tank (27° C., 130 rpm, 50% dissolvedoxygen for 48 hours).

3. Recombinant Protein Purification

Recombinant proteins can be purified from E. coli and/or insect cellsusing a variety of standard chromatography methods. Briefly, cells arelysed using sonication or detergents. The insoluble material is pelletedby centrifugation at 10,000×g for 15 minutes. The supernatant is appliedto an appropriate affinity column. For example, His-tagged proteins areseparated using a pre-packed chelating sepharose column (Pharmacia) orGST-tagged proteins are separated using a glutathione sepharose column(Pharmacia). After using the affinity column, proteins are furtherseparated using various techniques, such as ion exchange chromatography(columns from Pharmacia) to separate on the basis of electrical chargeor size exclusion chromatography (columns from Tosohaas) to separate onthe basis of molecular weight, size, and shape.

Expression and purification of the protein can also be achieved usingeither a mammalian cell expression system or an insect cell expressionsystem. The pUB6/V5-His vector system (Invitrogen, CA) can be used toexpress cDNA in CHO cells. The vector contains the selectable bsd gene,multiple cloning sites, the promoter/enhancer sequence from the humanubiquitin C gene, a C-terminal V5 epitope for antibody detection withanti-V5 antibodies, and a C-terminal polyhistidine (6×His) sequence forrapid purification on PROBOND resin (Invitrogen, CA). Transformed cellsare selected on media containing blasticidin.

Spodoptera frugiperda (Sf9) insect cells are infected with recombinantAutographica californica nuclear polyhedrosis virus (baculovirus). Thepolyhedrin gene is replaced with the cDNA by homologous recombinationand the polyhedrin promoter drives cDNA transcription. The protein issynthesized as a fusion protein with 6×His which enables purification asdescribed above. Purified proteins can be used to produce antibodies.

4. Chemical Synthesis of Proteins

Proteins or portions thereof can be produced not only by recombinantmethods (such as described above), but also by using chemical methodswell known in the art. Solid phase peptide synthesis can be carried outin a batchwise or continuous flow process which sequentially addsα-amino- and side chain-protected amino acid residues to an insolublepolymeric support via a linker group. A linker group such asmethylamine-derivatized polyethylene glycol is attached topoly(styrene-co-divinylbenzene) to form the support resin. The aminoacid residues are N-a-protected by acid labile Boc (t-butyloxycarbonyl)or base-labile Fmoc (9-fluorenylmethoxycarbonyl) groups. The carboxylgroup of the protected amino acid is coupled to the amine of the linkergroup to anchor the residue to the solid phase support resin.Trifluoroacetic acid or piperidine are used to remove the protectinggroup in the case of Boc or Fmoc, respectively. Each additional aminoacid is added to the anchored residue using a coupling agent orpre-activated amino acid derivative, and the resin is washed. Thefull-length peptide is synthesized by sequential deprotection, couplingof derivitized amino acids, and washing with dichloromethane and/orN,N-dimethylformamide. The peptide is cleaved between the peptidecarboxy terminus and the linker group to yield a peptide acid or amide.(Novabiochem 1997/98 Catalog and Peptide Synthesis Handbook, San DiegoCalif. pp. S1-S20).

Automated synthesis can also be carried out on machines such as the 431Apeptide synthesizer (Applied Bio systems, Foster City, Calif.). Aprotein or portion thereof can be purified by preparative highperformance liquid chromatography and its composition confirmed by aminoacid analysis or by sequencing (Creighton, 1984, Proteins, Structuresand Molecular Properties, WH Freeman, New York N.Y.).

5. Antibody Production

Polyclonal Antibodies

Polyclonal antibodies against recombinant proteins can be raised inrabbits (Green Mountain Antibodies, Burlington, Vt.). Briefly, two NewZealand rabbits are immunized with 0.1 mg of antigen in completeFreund's adjuvant. Subsequent immunizations are carried out using 0.05mg of antigen in incomplete Freund's adjuvant at days 14, 21, and 49.Bleeds are collected and screened for recognition of the antigen bysolid phase ELISA and Western blot analysis. The IgG fraction isseparated by centrifugation at 20,000×g for 20 minutes followed by a 50%ammonium sulfate cut. The pelleted protein is resuspended in 5 mM Trisand separated by ion exchange chromatography. Fractions are pooled basedon IgG content. Antigen-specific antibody is affinity purified usingPierce AminoLink resin coupled to the appropriate antigen.

Isolation of Antibody Fragments Directed Against a Protein Target from aLibrary of scFvs

Naturally occurring V-genes isolated from human PBLs can be constructedinto a library of antibody fragments which contain reactivities againsta target protein to which the donor may or may not have been exposed(see, for example, U.S. Pat. No. 5,885,793, incorporated herein byreference in its entirety).

Rescue of the library: A library of scFvs is constructed from the RNA ofhuman PBLs, as described in PCT publication WO 92/01047. To rescue phagedisplaying antibody fragments, approximately 10⁹ E. coli harboring thephagemid are used to inoculate 50 ml of 2×TY containing 1% glucose and100 μg/ml of ampicillin (2×TY-AMP-GLU) and grown to an O.D. of 0.8 withshaking. Five ml of this culture is used to innoculate 50 ml of2×TY-AMP-GLU, 2×10⁸ TU of delta gene 3 helper (M13 delta gene III, seePCT publication WO 92/01047) are added and the culture incubated at 37°C. for 45 minutes without shaking and then at 37° C. for 45 minutes withshaking. The culture is centrifuged at 4000 rpm. for 10 min. and thepellet resuspended in 2 liters of 2×TY containing 100 μg/ml ampicillinand 50 μg/ml kanamycin and grown overnight. Phage are prepared asdescribed in PCT publication WO 92/01047.

Preparation of M13 delta gene III: M13 delta gene III helper phage doesnot encode gene III protein, hence the phage(mid) displaying antibodyfragments have a greater avidity of binding to antigen. Infectious M13delta gene III particles are made by growing the helper phage in cellsharboring a pUC19 derivative supplying the wild type gene III proteinduring phage morphogenesis. The culture is incubated for 1 hour at 37°C. without shaking and then for a further hour at 37° C. with shaking.Cells are spun down (IEC-Centra 8,400 rpm for 10 min), resuspended in300 ml 2×TY broth containing 100 μg ampicillin/ml and 25 μg kanamycin/ml(2×TY-AMP-KAN) and grown overnight, shaking at 37° C. Phage particlesare purified and concentrated from the culture medium by twoPEG-precipitations (Sambrook et al., 2001, Molecular Cloning: ALaboratory Manual. 3rd. ed., Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y.), resuspended in 2 ml PBS and passed through a 0.45μm filter (Minisart NML; Sartorius) to give a final concentration ofapproximately 10¹³ transducing units/ml (ampicillin-resistant clones).

Panning of the library: Immunotubes (Nunc) are coated overnight in PBSwith 4 ml of either 100 μg/ml or 10 μg/ml of a protein target ofinterest. Tubes are blocked with 2% Marvel-PBS for 2 hours at 37° C. andthen washed 3 times in PBS. Approximately 10¹³ TU of phage is applied tothe tube and incubated for 30 minutes at room temperature tumbling on anover-and-under turntable and then left to stand for another 1.5 hours.Tubes are washed 10 times with PBS 0.1% Tween-20 and 10 times with PBS.Phage are eluted by adding 1 ml of 100 mM triethylamine and rotating 15minutes on an under-and-over turntable after which the solution isimmediately neutralized with 0.5 ml of 1.0 M Tris-HCl, pH 7.4. Phagesare then used to infect 10 ml of mid-log E. coli TG1 by incubatingeluted phage with bacteria for 30 minutes at 37° C. The E. coli are thenplated on TYE plates containing 1% glucose and 100 μg/ml ampicillin. Theresulting bacterial library is then rescued with delta gene 3 helperphage as described above to prepare phage for a subsequent round ofselection. This process is then repeated for a total of 4 rounds ofaffinity purification with tube-washing increased to 20 times with PBS,0.1% Tween-20 and 20 times with PBS for rounds 3 and 4.

Characterization of binders: Eluted phage from the 3rd and 4th rounds ofselection are used to infect E. coli HB 2151 and soluble scFv isproduced (Marks et al., 1991, J. Mol. Biol. 222: 581-597) from singlecolonies for assay. ELISAs are performed with microtitre plates coatedwith either 10 μg/ml of the protein target of interest in 50 mMbicarbonate pH 9.6. Clones positive in ELISA are further characterizedby PCR fingerprinting (see, e.g., PCT publication WO 92/01047) and thenby sequence analysis.

Monoclonal Antibodies

a) Materials:

1. Complete Media No Sera (CMNS) for washing of the myeloma and spleencells; Hybridoma medium CM-HAT (Cell Mab (BD), 10% FBS (or HS); 5%Origen HCF (hybridoma cloning factor) containing 4 mM L-glutamine andantibiotics) to be used for plating hybridomas after the fusion.

2. Hybridoma medium CM-HT (no aminopterin) (Cell Mab (BD), 10% FBS 5%Origen HCF containing 4 mM L-glutamine and antibiotics) to be used forfusion maintenance is stored in the refrigerator at 4-6° C. The fusionsare fed on days 4, 8, and 12, and subsequent passages. Inactivated andpre-filtered commercial fetal bovine serum (FBS) or horse serum (HS) arethawed and stored in the refrigerator at 4° C. and is pretested formyeloma growth from single cells prior to use.

3. The L-glutamine (200 mM, 100× solution), which is stored at −20° C.,is thawed and warmed until completely in solution. The L-glutamine isdispensed into media to supplement growth. L-glutamine is added to 2 mMfor myelomas and 4 mM for hybridoma media. Further, the penicillin,streptomycin, amphotericin (antibacterial-antifungal stored at −20° C.)is thawed and added to Cell Mab Media to 1%.

4. Myeloma growth media is Cell Mab Media (Cell Mab Media, QuantumYield, from BD, which is stored in the refrigerator at 4° C. in thedark), to which is added L-glutamine to 2 mM and antibiotic/antimycoticsolution to 1% and is called CMNS.

5. One bottle of PEG 1500 in Hepes (Roche, N.J.) is prepared.

6. 8-Azaguanine is stored as the dried powder supplied by SIGMA at −700°C. until needed. One vial/500 ml of media is reconstituted and theentire contents are added to 500 ml media (e.g., 2 vials/liter).

7. Myeloma Media is CM which has 10% FBS (or HS) and 8-Aza (1×) storedin the refrigerator at 4° C.

8. Clonal cell medium D (Stemcell, Vancouver) contains HAT and methylcellulose for semi-solid direct cloning from the fusion. This comes in90 ml bottles with a CoA and is melted at 37° C. in a waterbath in themorning of the day of the fusion. The cap is loosened and the bottle isleft in a CO₂ incubator to sufficiently gas the medium D and bring thepH down.

9. Hybridoma supplements HT [hypoxanthine, thymidine] to be used inmedium for the section of hybridomas and maintenace of hybridomasthrough the cloning stages, respectively.

10. Origen HCF can be obtained directly from Igen and is a cellsupernatant produced from a macrophage-like cell-line. It can be thawedand aliqouted to 15 ml tubes at 5 ml per tube and stored frozen at −20°C. Positive hybridomas are fed HCF through the first subcloning and aregradually weaned (individual hybridomas can continue to be supplemented,as needed). This and other additives are typically more effective inpromoting new hybridoma growth than conventional feeder layers.

b) Procedure:

To generate monoclonal antibodies, mice are immunized with 5-50 μg ofantigen, either intra-peritoneally (i.p.) or by intravenous injection inthe tail vein (i.v.). The antigen used can be a recombinant targetprotein of interest, for example. The primary immunization takes placetwo months prior to the harvesting of splenocytes from the mouse, andthe immunization is typically boosted by i.v. injection of 5-50 μg ofantigen every two weeks. At least one week prior to the expected fusiondate, a fresh vial of myeloma cells is thawed and cultured. Severalflasks of different densities can be maintained so that a culture at theoptimum density is ensured at the time of fusion. An optimum density canbe 3-6×10⁵ cells/ml, for example. 2-5 days before the scheduled fusion,a final immunization of approximately 5 μg of antigen in PBS isadministered (either i.p. or i.v).

Myeloma cells are washed with 30 ml serum free media by centrifugationat 500 g at 4° C. for 5 minutes. Viable cell density is determined inresuspended cells using hemocytometry and vital stains. Cellsresuspended in complete growth medium are stored at 37° C. during thepreparation of splenocytes. Meanwhile, to test aminopterin sensitivity,1×10⁶ myeloma cells are transferred to a 15 ml conical tube andcentrifuged at 500 g at 4° C. for 5 minutes. The resulting pellet isresuspended in 15 ml of HAT media and cells plated at 2 drops/well on a96-well plate.

To prepare splenocytes from immunized mice, the animals are euthanisedand submerged in 70% ethanol. Under sterile conditions, the spleen issurgically removed and placed in 10 ml of RPMI medium supplemented with20% fetal calf serum in a petri dish. Cells are extricated from thespleen by infusing the organ with medium >50 times using a 21 g syringe.

Cells are harvested and washed by centrifugation (at 500 g at 4° C. for5 minutes) with 30 ml of medium. Cells are resuspended in 10 ml ofmedium and the density of viable cells determined by hemocytometry usingvital stains. The splenocytes are mixed with myeloma cells at a ratio of5:1 (spleen cells: myeloma cells). Both the myeloma and spleen cells arewashed twice more with 30 ml of RPMI-CMNS, and the cells are spun at 800rpm for 12 minutes.

Supernatant is removed and cells are resuspended in 5 ml of RPMI-CMNSand are pooled to fill volume to 30 ml and spun down as before. Then,the pellet is broken up by gently tapping on the flow hood surface andresuspending in 1 ml of BMB REG1500 (prewarmed to 37° C.) dropwise witha 1 cc needle over 1 minute.

RPMI-CMNS to the PEG cells and RPMI-CMNS are added to slowly dilute outthe PEG. Cells are centrifuged and diluted in 5 ml of Complete media and95 ml of Clonacell Medium D (HAT) media (with 5 ml of HCF). The cellsare plated out 10 ml per small petri plate.

Myeloma/HAT control is prepared as follows: dilute about 1000 P3X63Ag8.653 myeloma cells into 1 ml of medium D and transfer into a singlewell of a 24-well plate. Plates are placed in an incubator, with twoplates inside of a large petri plate, with an additional petri platefull of distilled water, for 10-18 days under 5% CO₂ overlay at 37° C.Clones are picked from semisolid agarose into 96-well plates containing150-200 μl of CM-HT. Supernatants are screened 4 days later in ELISA,and positive clones are moved up to 24-well plates. Heavy growthrequires changing of the media at day 8 (+/−150 ml). The HCF can befurther decreased to 0.5% (gradually −2%, then 1%, then 0.5%) in thecloning plates.

6. Liquid Chromatography and Mass Spectrometry (LC/MS)

For LC/MS analysis, proteins are reduced in 2.5 mM DTT for 1 hour at 37°C., and alkylated with ICAT™ reagent according to the proceduresrecommended by the manufacturer (Applied Biosystems, Framingham, Mass.).The reaction is quenched by adding excess DTT. Proteins are digestedusing sequencing grade modified trypsin overnight at 37° C. followed bydesalting using 3 cc Oasis HLB solid phase extraction columns (Waters,Milford, Mass.) and vacuum drying. Cysteine-containing peptides arepurified by avidin column (Applied Biosystems, Framingham, Mass.). Thepeptides are reconstituted in buffer A (0.1% formic acid in water) andseparated over a C18 monomeric column (150 mm, 150 μm i.d., Grace Vydac238EV5, 5 μm) at a flow rate of 1.5 μl/min with a trap column. Peptidesare eluted from the column using a gradient, 3%-30% buffer B (0.1%formic acid in 90% acetonitrile) in 215 mM, 30%-90% buffer B in 30 min.Eluted peptides are analyzed using an online QSTAR XL system (MDS/Sciex,Toronto, ON). Peptide ion peaks from the map are automatically detectedwith RESPEC™ (PPL Inc., UK).

The sequence-composition of peptides detected, for example, at higherlevels in disease samples (or drug-resistant samples) relative toadjacent normal tissue (or drug-sensitive samples) can be resolvedthrough tandem mass spectrometry and database analysis. For dataanalysis, peptide ion peaks of LC/MS maps from normal and diseasesamples can be aligned based on mass to charge ratio (m/z), retentiontime (Rt), and charge state (z). The list of aligned peptide ions isloaded into Spotfire™ (Spotfire Inc. Somerville, Mass.). Intensities canbe normalized before further differential analysis between disease andnormal samples. Differentially expressed ions are manually verifiedbefore LC-MS/MS-based peptide sequencing and database searching forprotein/protein identification.

For intensity normalization and expression analysis, a heat map can beconstructed by sorting the rows by the ratio of the mean intensity inthe disease samples to the mean intensity of the normal samples. Rowsare included if there is at least one MS/MS identification of an ion inthe row. The display colors are determined for each row separately byassigning black to the median intensity in the row, green to the lowestintensity in the row, and red to the highest intensity.

Using a mass spectrometry procedure such as this, a comprehensiveanalysis of proteins differentially expressed by disease cells (or drugresistant cells, for example) compared with normal cells (or cellsresponsive/sensitive to a drug, for example) can be carried out.

7. mRNA Expression Analysis

Expression of target mRNA can be quantitated by RT-PCR using TaqMan®technology. The Taqman® system couples a 5′ fluorogenic nuclease assaywith PCR for real-time quantitation. A probe is used to monitor theformation of the amplification product.

Total RNA can be isolated from disease model cell lines using an RNEasyKit® (Qiagen, Valencia, Calif.) with DNase treatment (per themanufacturer's instructions). Normal human tissue RNAs can be acquiredfrom commercial vendors (e.g., Ambion, Austin, Tex.; Stratagene, LaJolla, Calif.; BioChain Institute, Newington, N.H.), as well as RNAsfrom matched disease/normal tissues.

Target transcript sequences can be identified for differentiallyexpressed peptides by database searching using a search algorithm suchas BLAST. TaqMan® assays (PCR primer/probe sets) specific for thosetranscripts can be obtained from Applied Biosystems (AB) as part of theAssays on Demand™ product line or by custom design through the AB Assaysby Design^(SM) service. If desired, the assays can be designed to spanexon-exon borders so as not to amplify genomic DNA.

RT-PCR can be accomplished using AmpliTaq Gold® and MultiScribe™ reversetranscriptase in the One Step RT-PCR Master Mix reagent kit (AB)(according to the manufacturer's instructions). Probe and primerconcentrations are 250 nM and 900 nM, respectively, in a 15 μl reaction.For each experiment, a master mix of the above components is made andaliquoted into each optical reaction well. Eight nanograms of total RNAis used as template. Quantitative RT-PCR can be performed using the ABIPrism® 7900HT Sequence Detection System (SDS). The following cyclingparameters are used: 48° C. for 30 min. for one cycle; 95° C. for 10 minfor one cycle; and 95° C. for 15 sec, 60° C. for 1 min. for 40 cycles.

SDS software can be utilized to calculate the threshold cycle (C_(T))for each reaction, and C_(T) values are used to quantitate the relativeamount of starting template in the reaction. The C_(T) values for eachset of reactions can be averaged for all subsequent calculations

Data can be analyzed to determine estimated copy number per cell. Geneexpression can be quantitated relative to 18S rRNA expression and copynumber estimated assuming 5×10⁶ copies of 18S rRNA per cell.Alternatively, data can be analyzed for fold difference in expressionusing an endogenous control for normalization and expressed relative toa normal tissue or normal cell line reference. The choice of endogenouscontrol can be determined empirically by testing various candidatesagainst the cell line and tissue RNA panels and selecting the one withthe least variation in expression. Relative changes in expression can bequantitated using the 2^(−ΔΔCT) method (Livak et al., 2001, Methods 25:402-408; User bulletin #2: ABI Prism 7700 Sequence Detection System).Alternatively, total RNA can be quantitated using a RiboGreen RNAQuantitation Kit according to manufacturer's instructions and thepercentage mRNA expression calculated using total RNA for normalization.Percentage knockdown can then be calculated relative to a no additioncontrol.

8. Flow Cytometry (FACS) Analysis

Flow cytometry is interchangeably referred to as fluorescence-activatedcell sorting (FACS). Quantitative flow cytometry can be used to comparethe level of expression of a protein on disease cells to the level foundon normal cells, for example.

Expression levels of a target protein on primary tissue samples can bequantified using the Quantum Simply Cellular System (Bangs Laboratories,Fishers, Ind.) and a target-specific antibody. Normal adjacent anddisease tissues can be processed into single cell suspensions, asdescribed above, which can be stained for various markers (e.g., theepithelial marker EpCam) and the target-specific antibody. At least0.5×10⁶ cells are typically used for each analysis. Cells are washedonce with Flow Staining Buffer (0.5% BSA, 0.05% NaN3 in D-PBS). To thecells, 20 μl of each target-specific antibody are added. An additional 5μl of anti-EpCam antibody conjugated to APC can be added when unsortedcells are used. Cells are incubated with antibodies for 30 minutes at 4°C. Cells are washed once with Flow Staining Buffer and either analyzedimmediately on an LSR flow cytometry apparatus or fixed in 1%formaldehyde and stored at 4° C. until LSR analysis. Antibodies used todetect a target can be PE-conjugated. PE-conjugated mouse IgG1k can usedas an isotype control antibody. Cells are analyzed by flow cytometry andepitope copy number and the percentage of viable epithelial cellspositive for target expression can be measured. Cell numbers andviability can be determined by PI exclusion (GUAVA) for cells isolatedfrom both normal and disease tissue. Standard curve and samples can beanalyzed on a LSR I (BDBiosciences, San Jose Calif.) flow cytometer.Antibody binding capacity for each lineage population can be calculatedusing geometric means and linear regression.

Expression levels of a target protein can be quantified in cell lineswith QIFIKIT flow cytometric indirect immunofluorescence assay (DakoA/S) using a primary antibody to the target. Briefly, cells are detachedwith versene or trypsin and washed once with complete media and thenPBS. 5×10⁵ cells/sample are incubated with saturating concentration (10μg/ml) of primary antibody for 60 minutes at 4° C. After washes, aFITC-conjugated secondary antibody (1:50 dilution) is added for 45minutes at 4° C. QIFIKIT standard beads are simultaneously labeled withthe secondary antibody. Binding of antibodies is analyzed by flowcytometry and specific antigen density is calculated by subtractingbackground antibody equivalent from antibody-binding capacity based on astandard curve of log mean fluorescence intensity versus log antigenbinding capacity.

Cells can also be prepared for flow cytometry analysis (as well as othertypes of analysis) as follows: cells are incubated with 1:100 dilutionof BrdU in culturing media for 2-4 hours (BrdU Flow Kit, cat #559619 BDBiosciences). Cells are washed 3 times with D-PBS and disassociated fromthe flask with versene. Cell numbers and viability can be determined byPI exclusion (GUAVA). Cells are washed once with Flow Staining Buffer(0.5% BSA, 0.05% NaN₃ in D-PBS). Cells are incubated with 400 μl ofCytofix/Cytoperm Buffer (BrdU Flow Kit, BD Biosciences) for 15-30minutes at 4° C. Cells are washed once with Flow Staining Buffer andresuspended in 400 μl Cytoperm Plus Buffer (BrdU Flow Kit BDBiosciences). Cells are incubated for 10 minutes at 4° C. and washedonce with 1× Perm/Wash Buffer (BrdU Flow Kit, BD Biosciences). Cells areincubated for 1 hour at 37° C. protected from light in DNAse solution(BrdU Flow Kit, BD Biosciences). Cells are washed once with 1× Perm/WashBuffer and incubated for 20 min at room temperature with anti-BrdUFITC-conjugated antibody (BrdU Flow Kit, BD Biosciences), PE-conjugatedactive caspase 3 (BD Biosciences cat #550821), and PE mouse IgG2Bisotype control. Cells are washed once with 1× Perm/Wash Buffer andresuspended in DAPI for LSR flow cytometry analysis.

9. Immunohistochemistry (IHC)

IHC of Tissue Sections

Paraffin embedded, fixed tissue sections (e.g., from disease tissuesamples such as solid tumors or other cancer tissues) can be obtainedfrom a panel of normal tissues as well as tumor (or other disease)samples with matched normal adjacent tissues, along with replicatesections (if desired). For example, for an initial survery of targetexpression, a panel of common cancer formalin-fixed paraffin-embedded(FFPE) tissue microarrays (TMAs) can be used for analysis, and such TMAscan be obtained from commercial sources (TriStar, Rockville, Md.;USBiomax, Rockville, Md.; Imgenex, San Diego, Calif.; Petagen/Abxis,Seoul, Korea). Sections can be stained with hemotoxylin and eosin andhistologically examined to ensure adequate representation of cell typesin each tissue section.

An identical set of tissues can be obtained from frozen sections for usein those instances where it is not possible to generate antibodies thatare suitable for fixed sections. Frozen tissues do not require anantigen retrieval step.

Paraffin Fixed Tissue Sections

An exemplary protocol for hemotoxylin and eosin staining of paraffinembedded, fixed tissue sections is as follows. Sections aredeparaffinized in three changes of xylene or xylene substitute for 2-5minutes each. Sections are rinsed in two changes of absolute alcohol for1-2 minutes each, in 95% alcohol for 1 minute, followed by 80% alcoholfor 1 minute. Slides are washed in running water and stained in Gillsolution 3 hemotoxylin for 3-5 minutes. Following a vigorous wash inrunning water for 1 minute, sections are stained in Scott's solution for2 minutes. Sections are washed for 1 minute in running water and thencounterstained in eosin solution for 2-3 minutes, depending upon thedesired staining intensity. Following a brief wash in 95% alcohol,sections are dehydrated in three changes of absolute alcohol for 1minute each and three changes of xylene or xylene substitute for 1-2minutes each. Slides are coverslipped and stored for analysis.

Optimization of Antibody Staining

For each antibody, a positive and negative control sample can begenerated using data from ICAT analysis of disease cell lines ortissues. Cells can be selected that are known to express low levels of aparticular target as determined from the ICAT data, and this cell linecan be used as a reference normal control. Similarly, a disease cellline that is determined to over-express the target can also be selected.

Antigen Retrieval

Sections are deparaffinized and rehydrated by washing 3 times for 5minutes in xylene, two times for 5 minutes in 100% ethanol, two timesfor 5 minutes in 95% ethanol, and once for 5 minutes in 80% ethanol.Sections are then placed in endogenous blocking solution (methanol+2%hydrogen peroxide) and incubated for 20 minutes at room temperature.Sections are rinsed twice for 5 minutes each in deionized water andtwice for 5 minutes in phosphate buffered saline (PBS), pH 7.4.

Alternatively, where necessary, sections are de-parrafinized by HighEnergy Antigen Retrieval as follows: sections are washed three times for5 minutes in xylene, two times for 5 minutes in 100% ethanol, two timesfor 5 minutes in 95% ethanol, and once for 5 minutes in 80% ethanol.Sections are placed in a Coplin jar with dilute antigen retrievalsolution (10 mM citrate acid, pH 6). The Coplin jar containing slides isplaced in a vessel filled with water and microwaved on high for 2-3minutes (700 watt oven). Following cooling for 2-3 minutes, steps 3 and4 are repeated four times (depending on the tissue), followed by coolingfor 20 minutes at room temperature. Sections are then rinsed indeionized water (two times for 5 minutes), placed in modified endogenousoxidation blocking solution (PBS+2% hydrogen peroxide), and rinsed for 5minutes in PBS.

Alternatively, formalin fixed paraffin embedded tissues can bedeparaffinized and processed for antigen retrieval using theEZ-retriever system (BioGenex, San Ramon, Calif.). EZ-antigen Retrievalcommon solution is used for deparaffinization and EZ-retrievalcitrate-based buffer used for antigen retrieval. Samples are pre-blockedwith non-serum protein block (Dako A/S, Glostrup, Denmark) for 15 min.Primary antibodies (at 2.5-5.0 μg/ml, for example) are incubatedovernight at room temperature. Envision Plus system HRP (Dako A/S) isused for detection with diaminobenzidine (DAB) as substrate forhorseradish peroxidase.

Blocking and Staining

Sections are blocked with PBS/1% bovine serum albumin (PBA) for 1 hourat room temperature followed by incubation in normal serum diluted inPBA (2%) for 30 minutes at room temperature to reduce non-specificbinding of antibody. Incubations are performed in a sealed humiditychamber to prevent air-drying of the tissue sections. The choice ofblocking serum is typically the same as the species of the biotinylatedsecondary antibody. Excess antibody is gently removed by shaking andsections covered with primary antibody diluted in PBA and incubatedeither at room temperature for 1 hour or overnight at 4° C. (care istaken that the sections do not touch during incubation). Sections arerinsed twice for 5 minutes in PBS, shaking gently. Excess PBS is removedby gently shaking. The sections are covered with diluted biotinylatedsecondary antibody in PBA and incubated for 30 minutes to 1 hour at roomtemperature in the humidity chamber. If using a monoclonal primaryantibody, addition of 2% rat serum can be used to decrease thebackground on rat tissue sections. Following incubation, sections arerinsed twice for 5 minutes in PBS, shaking gently. Excess PBS is removedand sections incubated for 1 hour at room temperature in Vectastain ABCreagent (as per kit instructions). The lid of the humidity chamber issecured during all incubations to ensure a moist environment. Sectionsare rinsed twice for 5 minutes in PBS, shaking gently.

Developing and Counterstaining

Sections are incubated for 2 minutes in peroxidase substrate solutionthat is made up immediately prior to use as follows: 10 mgdiaminobenzidine (DAB) dissolved in 10 ml of 50 mM sodium phosphatebuffer, pH 7.4; 12.5 microliters 3% CoCl₂/NiCl₂ in deionized water; and1.25 microliters hydrogen peroxide.

Slides are rinsed well three times for 10 minutes in deionized water andcounterstained with 0.01% Light Green acidified with 0.01% acetic acidfor 1-2 minutes, depending on the desired intensity of counterstain.

Slides are rinsed three times for 5 minutes with deionized water anddehydrated two times for 2 minutes in 95% ethanol; two times for 2minutes in 100% ethanol; and two times for 2 minutes in xylene. Stainedslides are mounted for visualization by microscopy.

Slides are scored manually using a microscope such as the Zeiss Axiovert200M microscope (Carl Zeiss Microimaging, Thornwood, N.Y.).Representative images are acquired using 40× objective (400×magnification).

IHC Staining of Frozen Tissue Sections

For IHC staining of frozen tissue sections, fresh tissues are embeddedin OCT in plastic mold, without trapping air bubbles surrounding thetissue. Tissues are frozen by setting the mold on top of liquid nitrogenuntil 70-80% of the block turns white at which point the mold is placedon dry ice. The frozen blocks are stored at −80° C. Blocks are sectionedwith a cryostat with care taken to avoid warming to greater than −10° C.Initially, the block is equilibrated in the cryostat for about 5 minutesand 6-10 mm sections are cut sequentially. Sections are allowed to dryfor at least 30 minutes at room temperature. Following drying, tissuesare stored at 4° C. for short term and −80° C. for long term storage.

Sections are fixed by immersing in an acetone jar for 1-2 minutes atroom temperature, followed by drying at room temperature. Primaryantibody is added (diluted in 0.05 M Tris-saline [0.05 M Tris, 0.15 MNaCl, pH 7.4], 2.5% serum) directly to the sections by covering thesection dropwise to cover the tissue entirely. Binding is carried out byincubation in a chamber for 1 hour at room temperature. Without lettingthe sections dry out, the secondary antibody (diluted inTris-saline/2.5% serum) is added in a similar manner to the primaryantibody and incubated as before (at least 45 minutes).

Following incubation, the sections are washed gently in Tris-saline for3-5 minutes and then in Tris-saline/2.5% serum for another 3-5 minutes.If a biotinylated primary antibody is used, in place of the secondaryantibody incubation, slides are covered with 100 μl of diluted alkalinephosphatase conjugated streptavidin, incubated for 30 minutes at roomtemperature and washed as above. Sections are incubated with alkalinephosphatase substrate (1 mg/ml Fast Violet; 0.2 mg/ml Napthol AS-MXphosphate in Tris-Saline pH 8.5) for 10-20 minutes until the desiredpositive staining is achieved at which point the reaction is stopped bywashing twice with Tris-saline. Slides are counter-stained with Mayer'shematoxylin for 30 seconds and washed with tap water for 2-5 minutes.Sections are mounted with Mount coverslips and mounting media.

10. RNAi Assays in Cell Lines

RNAi Transfections

Expression of a target can be knocked down by transfection with smallinterfering RNA (siRNA) to that target. Synthetic siRNA oligonucleotidescan be obtained from Dharmacon (Lafayette, Colo.) or Qiagen (Valencia,Calif.). For siRNA transfection, cells (e.g., disease cells) can beseeded into 96 well tissue culture plates at a density of 2,500 cellsper well 24 hours before transfection. Culture medium is removed and 50μl of reaction mix containing siRNA (final concentration 1 to 100 nM)and 0.4 μl of DharmaFECT4 (Dharmacon, Lafayette, Colo.) diluted inOpti-MEM is added to each well. An equal volume of complete mediumfollows and the cells are then incubated at 5% CO₂ at 37° C. for 1 to 4days.

Alternatively, in the initial screening phase, RNAi can be performedusing 100 nM (final) of Smartpools (Dharmacon, Lafayette, Colo.), poolof 4- for Silencing siRNA duplexes (Qiagen, Valencia, Calif.), ornon-targeting negative control siRNA (Dharmacon or Qiagen). In thebreakout phase, each individual duplex is used at 100 nM (final). In thetitration phase, individual duplex is used at 0.1-100 nM (final).Transient transfections are carried out using either Lipofectamine 2000from Invitrogen (Carlsbad, Calif.) or GeneSilencer from Gene TherapySystems (San Diego, Calif.) (see below). One day after transfections,total RNA is isolated using the RNeasy 96 Kit (Qiagen) according tomanufacturer's instructions and expression of mRNA is quantitated usingTaqMan technology. Apoptosis and cell proliferation assays can beperformed daily using Apop-one homogeneous caspase-3/7 kit and AlamarBlue or CellTiter 96 AQueous One Solution Cell Proliferation Assays (seebelow).

RNAi Transfections—Lipofectamine 2000 and GeneSilencer

Transient RNAi transfections can be carried out using Lipofectamine 2000(Invitrogen, Carlsbad, Calif.) or GeneSilencer (Gene Therapy Systems,San Diego, Calif.), such as on sub-confluent disease cell lines, asdescribed elsewhere (Elbashir et al., 2001, Nature 411: 494-498; Caplenet al., 2001, Proc Natl Acad Sci USA 98: 9742-9747; Sharp, 2001, Genesand Development 15: 485-490). Synthetic RNA to a gene of interest ornon-targeting negative control siRNA are transfected using Lipofectamine2000 or GeneSilencer according to manufacturer's instructions. Cells areplated in 96-well plates in antibiotic-free medium. The next day, thetransfection reagent and siRNA are prepared for transfections asfollows.

0.1-100 nM siRNA is resuspended in 20-25 μl serum-free media in eachwell (with Plus for Lipofectamine 2000) and incubated at roomtemperature for 15 minutes. 0.1-1 μl of Lipofectamine 2000 or 1-1.5 μlof GeneSilencer is also resuspended in serum-free medium to a finalvolume of 20-25 μl per well. After incubation, the diluted siRNA andeither the Lipofectamine 2000 or the GeneSilencer are combined andincubated for 15 minutes (Lipofectamine 2000) or 5-20 minutes(GeneSilencer) at room temperature. Media is then removed from the cellsand the combined siRNA-Lipofectamine 2000 reagent or siRNA-GeneSilencerreagent is added to a final volume of 50 μl per well. After furtherincubation at 37° C. for 4 hours, 50 μl serum-containing medium is addedback to the cells. 1-4 days after transfection, expression of mRNA canbe quantitated by RT-PCR using TaqMan technology, and protein expressionlevels can be measured by flow cytometry. Apoptosis and proliferationassays can be performed daily using Apop-one homogeneous caspase-3/7 kitand Alamar Blue or CellTiter 96 AQueous One Solution Cell ProliferationAssays (see below).

mRNA and Protein Knockdowns

Knockdown of target mRNA levels can be monitored by Q-PCR one day aftersiRNA transfection by using a TaqMan® assay (Applied Biosystems, FosterCity, Calif.). RT-PCR is accomplished in a one-step reaction by usingM-MLV reverse transcriptase (Promega, Madison, Wis.) and AmpliTaq Gold®(ABI) and analyzed on the ABI Prism® 7900HT Sequence Detection System(ABI). Relative gene expression can be quantitated by the ΔΔCt method(User Bulletin #2, ABI) with 18S rRNA serving as the endogenous control.

Protein knockdown can be monitored by FACS four days after transfectionby using an antibody to the target. The samples can be run on a LSR flowcytometer (BD Biosciences, San Jose, Calif.) and live cells monitored byusing PI exclusion (50 μg/ml PI, 2.5 units/ml RNase A, 0.1% Triton X-100in D-PBS). The data can be analyzed using CellQuest software.

Cell Proliferation—Alamar Blue

Cell growth can be assessed four days after transfection by adding a1:10 dilution of Alamar blue reagent (Invitrogen, Carlsbad, Calif. orBiosource, Camarillo, Calif.) and incubated for 2 hours at 37° C.Analysis can be performed on a Spectrafluor Plus (Tecan, Durham, N.C.)set at excitation wavelength of 530 nm and emission wavelength of 595nm.

Cell Proliferation—MTS

Alternatively, cell proliferation assays can be performed using aCellTiter 96 AQueous One Solution Cell Proliferation Assay kit (Promega,Madison, Wis.). 200 of CellTiter 96 AQueous One Solution is added to1000 of culture medium. The plates are then incubated for 1-4 hours at37° C. in a humidified 5% CO₂ incubator. After incubation, the change inabsorbance is read at 490 nm.

Apoptosis

Apoptosis assays can be performed using the Apop-one homogeneouscaspase-3/7 kit (Promega, Madison, Wis.). Briefly, the caspase-3/7substrate is thawed to room temperature and diluted 1:100 with buffer.The diluted substrate is then added 1:1 to cells, control, or blank. Theplates are then placed on a plate shaker for 30 minutes to 18 hours at300-500 rpm. The fluorescence of each well is then measured using anexcitation wavelength of 485+/−20 nm and an emission wavelength of530+/−25 nm.

11. Antibody Assays in Cell Lines

Cytotoxicity Assays

Cytotoxicity can be measured using a Resazurin (Sigma, Mo.) dyereduction assay (McMillian et al., 2002, Cell Biol. Toxicol.18:157-173). Briefly, cells are plated at 1,000-5,500 cells/well in 96well plates, allowed to attach to the plates for 18 hours beforeaddition of fresh media with or without antibody. After 96-144 hours ofexposure to antibody, resazurin is added to cells to a finalconcentration of 50 μM. Cells are incubated for 2-6 hours depending ondye conversion of cell lines, and dye reduction is measured on a FusionHT fluorescent plate reader (Packard Instruments, Meridien, Conn.) withexcitation and emission wavelengths of 530 nm and 590 nm, respectively.The IC₅₀ value is defined here as the drug concentration that results in50% reduction in growth or viability as compared with untreated controlcultures.

Assays for Antibody-Dependent Cellular Cytotoxicity

Antibody-dependent cellular cytotoxicity (ADCC) assays can be carriedout as follows. Cultured disease cells (e.g., tumor cells) are labeledwith 100 μCi ⁵¹Cr for 1 hour (Livingston et al., 1997, Cancer Immunol.Immunother. 43, 324-330). After being washed three times with culturemedium, cells are resuspended at 10⁵/ml, and 100 μl/well are plated onto96-well round-bottom plates. A range of antibody concentrations areapplied to the wells, including an isotype control together with donorperipheral blood mononuclear cells that are plated at a 100:1 and 50:1ratio. After an 18 hour incubation at 37° C., supernatant (30 μl/well)is harvested and transferred onto Lumaplate 96 (Packard), dried, andread in a Packard Top-Count NXT γ counter. Spontaneous release isdetermined by cpm of disease cells incubated with medium and maximumrelease by cpm of disease cells plus 1% Triton X-100 (Sigma). Specificlysis is defined as: % specific lysis=[(experimental release−spontaneousrelease)/(maximum release−spontaneous release)]×100. The percent ADCC isexpressed as peak specific lysis postimmune subtracted by preimmunepercent specific lysis. A doubling of the ADCC to >20% can typically beconsidered significant.

Assays for Complement Dependent Cytotoxicity

Chromium release assays to assess complement dependent cytotoxicity(CDC) can be carried out as follows (Dickler et al., 1999, Clin. CancerRes. 5, 2773-2779). Cultured disease cells (e.g., tumor cells) arewashed in FCS-free media two times, resuspended in 500 μl of media, andincubated with 100 μCi ⁵¹Cr per 10 million cells for 2 hours at 37° C.The cells are then shaken every 15 min for 2 hours, washed 3 times inmedia to achieve a concentration of approximately 20,000 cells/well, andthen plated in round-bottom plates. The plates contain either 50 μlcells plus 50 μl monoclonal antibody, 50 μl cells plus serum (pre- andpost-therapy), or 50 μl cells plus mouse serum as a control. The platesare incubated in a cold room on a shaker for 45 min. Human complement ofa 1:5 dilution (resuspended in 1 ml of ice-cold water and diluted with3% human serum albumin) is added to each well at a volume of 100 μl.Control wells include those for maximum release of isotope in 10% TritonX-100 (Sigma) and for spontaneous release in the absence of complementwith medium alone. The plates are incubated for 2 hours at 37° C.,centrifuged for 3 min, and then 100 μl of supernatant is removed forradioactivity counting. The percentage of specific lysis is calculatedas follows: % cytotoxicity=[(experimental release−spontaneousrelease)/(maximum release−spontaneous release)]×100. A doubling of theCDC to >20% can typically be considered significant.

Cell Proliferation Assays

To measure cell proliferation, cells can be plated, grown and treated asfor the cytotoxicity assay (above) in 96 well plates. After 96-144 hoursof treatment, 0.5 μCi/well ³H-Thymidine (PerkinElmer, 6.7 Ci/mmol) isadded to cells and incubated for 4-6 hours at 37° C., 5% CO₂ in anincubator. To lyse cells, plates are frozen overnight at −20° C. andthen cell lysates are harvested using FilterMate (Packard Instrument,Meridien, Conn.) into 96 well filter plates. Radioactivity associatedwith cells is measured on a TopCount (Packard) scintillation counter.

Other cell assays (e.g., proliferation assays such as Alamar blue andMTS, and apoptosis assays) can be carried out using antibodies, asdescribed above for RNAi.

Testing of Function-Blocking Antibodies

For testing of function-blocking antibodies, sub-confluent disease celllines are serum-starved overnight. The next day, serum-containing mediais added back to the cells in the presence of 5-50 ng/ml offunction-blocking antibodies. After 2 or 5 days incubation at 37° C. 5%CO₂, antibody binding is examined by flow cytometry, and apoptosis andproliferation are measured.

Cell Invasion

Cell invasion assays can be performed using a 96-well cell invasionassay kit (Chemicon). After the cell invasion chamber plates areadjusted to room temperature, 100 μl serum-free media is added to theinterior of the inserts. 1-2 hours later, cell suspensions of 1×10⁶cells/ml are prepared. Media is then carefully removed from the insertsand 100 μl of prepared cells are added into the insert +/−0 to 50 ngfunction blocking antibodies. The cells are pre-incubated for 15 minutesat 37° C. before 150 μl of media containing 10% FBS is added to thelower chamber. The cells are then incubated for 48 hours at 37° C. Afterincubation, the cells from the top side of the insert are discarded andthe invasion chamber plates are then placed on a new 96-well feeder traycontaining 150 μl of pre-warmed cell detachment solution in the wells.The plates are incubated for 30 minutes at 37° C. and are periodicallyshaken. Lysis buffer/dye solution (4 μl CyQuant Dye/300 μl 4× lysisbuffer) is prepared and added to each well of dissociation buffer/cellson feeder tray. The plates are incubated for 15 minutes at roomtemperature before 150 μl is transferred to a new 96-well plate.Fluorescence of invading cells is then read at 480 nm excitation and 520nm emission.

Receptor Internalization

For quantification of receptor internalization, ELISA assays can beperformed essentially as described by Daunt et al. (Daunt et al., 1997,Mol. Pharmacol. 51, 711-720). Cell lines are plated at 6×10⁵ cells perin a 24-well tissue culture dishes that have previously been coated with0.1 mg/ml poly-L-lysine. The next day, the cells are washed once withPBS and incubated in DMEM at 37° C. for several minutes. Agonist to thecell surface target of interest is then added to the wells at apre-determined concentration in prewarmed DMEM. The cells are thenincubated for various times at 37° C. and reactions are stopped byremoving the media and fixing the cells in 3.7% formaldehyde/TBS for 5min at room temperature. The cells are then washed three times with TBSand nonspecific binding blocked with TBS containing 1% BSA for 45 min atroom temperature. The first antibody is added at a pre-determineddilution in TBS/BSA for 1 hr at room temperature. Three washes with TBSfollow, and cells are briefly reblocked for 15 min at room temperature.Incubation with goat anti-mouse conjugated alkaline phosphatase(Bio-Rad) diluted 1:1000 in TBS/BSA is carried out for 1 hr at roomtemperature. The cells are washed three times with TBS and acolorimetric alkaline phosphatase substrate is added. When the adequatecolor change is reached, 100 μl samples are taken for colorimetricreadings.

12. Treatment with Antibodies

Treatment of Disease Cells with Monoclonal Antibodies.

Disease cells (e.g., cancer cells), or cells such as NIH 3T3 cells thatexpress a target of interest, are seeded at a density of 4×10⁴ cells perwell in 96-well microtiter plates and allowed to adhere for 2 hours. Thecells are then treated with different concentrations of monoclonalantibody (Mab) specific for the protein target of interest, orirrelevant isotype matched (e.g., anti-rHuIFN-gamma) Mab, at 0.05, 0.5or 5.0 μg/ml. After a 72 hour incubation, the cell monolayers arestained with crystal violet dye for determination of relative percentviability (RPV) compared to control (untreated) cells. Each treatmentgroup can have replicates. Cell growth inhibition is monitored.

In Vivo Treatment with Monoclonal Antibodies.

NIH 3T3 cells transfected with either an expression plasmid thatexpresses the target of interest or a neo-DHFR vector are injected intonu/nu (athymic) mice subcutaneously at a dose of 10⁶ cells in 0.1 ml ofphosphate-buffered saline. On days 0, 1, 5, and every 4 days thereafter,100 μg (0.1 ml in PBS) of a Mab specific for the protein target ofinterest, or an irrelevant Mab, of the IgA2 subclass is injectedintraperitoneally. Disease progression (e.g., tumor occurrence and size)can be monitored for a one month period of treatment, for example.

13. Examples of Results from Experimental Validation

The Genbank protein accession numbers, and protein SEQ ID NOScorresponding to Table 1, for the four targets described below are asfollows: Angiopoietin-like 4 (ANGPTL4)=Q9BY76 (protein SEQ IDNOS:476-478 in Table 1), Tweety Homolog 3 (TTYH3)=NP_(—)079526(exemplary TTYH3 protein and encoding transcript sequences are providedin the sequence listing as SEQ ID NOS:1977-1978), CEACAM6(carcinoembryonic antigen-related cell adhesion molecule 6precursor)=P40199 or NP_(—)002474 (exemplary CEACAM6 protein andencoding transcript sequences are provided in the sequence listing asSEQ ID NOS:1975-1976), and protein FLJ11273=gi|20380426 (protein SEQ IDNOS:713-714 in Table 1).

Angiopoietin-Like 4 (ANGPTL4)

ANGPTL4 peptides were identified by mass spec to be over-expressed by36-fold in spheroid cells (i.e., cancer stem cells) compared withparental adherent cells from lung cancer cell line H1299.

See FIG. 1 for further results from experimental validation of ANGPTL4.

Tweety Homolog 3 (TTYH3)

A TTYH3 peptide was identified by mass spec as over-expressed by thefollowing ratios: 6.8-fold in spheroid cells (i.e., cancer stem cells)compared with differentiated cells from a colon cancer cell line(referred to as CBT026T), 9.9-fold in a colon tumor tissue sample, and6.5-fold in a liver cancer cell line.

See FIG. 2 for further results from experimental validation of TTYH3.

CEACAM6 (carcinoembryonic antigen-related cell adhesion molecule 6precursor)

CEACAM6 peptides were identified by mass spec as over-expressed by thefollowing ratios: 5.5-fold in spheroid cells (i.e., cancer stem cells)compared with differentiated cells from a colon cancer cell line(referred to as CBT026T), 9.4-49.0 fold in a colon tumor tissue sample,4.6-5.3 fold in a liver tumor tissue sample, and 9.1-24.0 fold in apancreatic cancer cell line.

See FIG. 3 for further results from experimental validation of CEACAM6.

Protein FLJ11273

An FLJ11273 peptide was identified by mass spec as over-expressed by5.5-fold in spheroid cells (i.e., cancer stem cells) compared withdifferentiated cells from a colon cancer cell line (referred to asCBT026T).

See FIG. 4 for further results from experimental validation of FLJ11273.

14. Examples of Results of RNAi Assays in Cancer Stem Cell Lines

RNAi knockdown of the prominin 1 (PROM1) target in colon cancer stemcell line CBT026T-HES induced apoptosis and inhibited cell proliferationin this cell line (prominin 1 is represented in Table 1 by protein SEQID NOS:411-413 and 700).

RNAi knockdown of the integrin beta-6 (ITGB6) target in the HT29 coloncancer stem cell line inhibited cell proliferation in this cell line(ITGB6 is represented in Table 1 by protein SEQ ID NOS:379-380).

All publications and patents mentioned in the above specification areherein incorporated by reference. Various modifications and variationsof the described methods and compositions of the invention will beapparent to those skilled in the art without departing from the scopeand spirit of the invention. Although the invention has been describedin connection with specific exemplary embodiments, it should beunderstood that the invention as claimed should not be unduly limited tosuch specific embodiments. Indeed, various modifications of theabove-described modes for carrying out the invention, which are obviousto those skilled in the field of molecular biology or related fields,are intended to be within the scope of the following claims.

TABLE 3 Subcellular Target Symbol IHC Apoptosis Proliferation MatrixLocation ITAV_HUMAN H1299_StemCell_Cys_Only Cell Surface SLC1A5 30%Ovarian Tumors Colon (++) CBT026_HES_Primary Cell Surface 40% ProstateTumors Pancreatic (++) 40% Met Panc Tumors Prostate (++) 20% Panc TumorsATP1B3 100% Glioblastoma Kidney (++) Pancreatic (+++) HT29_Stemcell CellSurface 80% NSC Lung Tumors Melanoma (++) Gastric (++)CBT026_HES_Primary 71% Panc Tumors Pancreatic (++) Kidney (++) 70%Breast Gastric (+) Lung (++) 70% Melanoma Melanoma (++) 70% MelanomaNode 67% Met Panc Tumors 70% Squ Lung 50% Colon 50% Ovary 38% LiverTumors 30% Gastric Tumors TSN1_HUMAN 50% Liver Tumors Colon (++)CBT026_T Cell Surface 40% Melanoma Node Gastric (++) CBT026_HES_Primary100% Pancreas Tumors Melanoma (++) 100% Met Pancreas Tumors Pancreatic(+) Prostate (+) TFR1_HUMAN CBT026_N Cell Surface CBT026_ HES_ PrimaryT4S3_HUMAN Pancreatic (++) CBT026_N Cell Surface CBT026_T M6PR Lung (++)HT29_Stemcell Cell Surface Pancreatic (++) CBT026_N Gastric (+)CBT026_HES_Primary BST2 Colon (++) Colon (++) CBT026_N Cell SurfaceLiver (++) Liver (++) RKO Sphere vs Lung (++) Lung (++) Adherent PNGFMelanoma (++) Melanoma (++) Ovary (+) CEACAM5 CBT026_N Cell SurfaceCBT026_T CBT026_HES_Primary ITGA6 100% Kidney Tumors Colon (++) Lung(+++) CBT026_N Cell Surface 80% NSC Lung Tumors Colon (++) CBT026_T 70%Squ Lung Tumors Gastric (++) CBT026_HES_Primary 67% Met Panc Tumors 60%Melanoma Node 50% Melanoma 50% Glioblastoma 40% Gastric Tumors 63% LiverTumors 25% Panc Tumors 20% Ovary Tumors 20% Colon Tumors ITGB4 40%Breast Tumors Colon (++) CBT026_N Cell Surface CBT026_TCBT026_HES_Primary C20orf3 80% Melanoma Colon (++) CBT026_N Cell Surface70% Melanoma Node Liver (++) CBT026_T 50% NHL, Lymph Node Lung (++)CBT026_HES_Primary 50% Colon Tumors RKO Sphere vs 50% Kidney TumorsAdherent PNGF 83% Glioblastoma 33% Met Panc Tumors 29% Panc Tumors 20%Prostate Tumors 25% Liver Tumors TACSTD1 CBT026_N Cell Surface CBT026_TCBT026_HES_Primary TM9SF3 90% Melanoma Pancreatic (++) HT29_StemcellCell Surface 50% Glioblastoma CBT026_N 40% Colon TumorsCNT026_HES_Primary 60% Melanoma Node 33% NHL Node 20% Prostate TumorsECE1 H1299_StemCell_Glyco Cell Surface CALR CBT026_N Cell Surface ICAM1RKO Sphere vs Cell Surface Adherent PNGF ADAM10 CBT026_N Cell SurfaceSYPL CBT026_ HES_Primary CBT026_T Cell Surface Melanoma Stemcell_EGT003DLMAN2 HT29_StemCell Cell Surface CBT026_N H1299_StemCell_Glyco ITM1Colon (++) Colon (++) HT29_StemCell Cell Surface CBT026_N CBT026_T MUC13100% Glioblastoma Pancreatic (++) Gastric (++) CBT026_N Cell Surface100% Colon Tumors Colon (+) CBT026_HES_Primary 30% Esoph Tumors 100%Liver Tumors 30% Melanoma Node 70% Pancreas Tumors 70% Melanoma 30%Gastric Tumors LTF Melanoma Secreted StemCell_EGT003D SLC5A1 33%Glioblastoma Colon (++) Colon (++) CBT026_N Cell Surface 100% ColonTumors Gastric (++) 90% Kidney Tumors Pancreatic (++) 88% Liver Tumors70% NSC Lung Tumors 20% Squ Lung Tumors 50% Melanoma Node 50% NHL Node86% Panc Tumors 100% Met Panc Tumors 50% Melanoma PROM1 60% Colon Lung(++) Colon (++) CBT026_N Cell Surface Lung (++) CBT026_TCBT026_HES_Primary NPC2 70% Breast Tumors Colon (++) Melanoma Secreted50% Colon Tumors Stemcell_EGT003D 70% NSC Lung Tumors 43% Panc Tumors66% Met Panc Tumors 20% Prostate Tumors CD68 Kidney (++) Liver (++) CSCOverlap Cell Surface Gastric (+) CBT026_N CTSB Melanoma StemCell_EGT003DSecreted CTSD Gastric (++) CBT026_N Liver (++) Melanoma SecretedStemcell_EGT003D OLFM4 70% Bladder Tumors CBT026_N Secreted 20% ColonTumors 100% Kidney Tumors 38% Liver Tumors 40% Melanoma Node 33% NHLNode 50% Gastric Tumors CDH17 CBT026_N Cell Surface CBT026_TCBT026_HES_Primary ITGB6 Colon (++) Colon (++) HT29_StemCell CellSurface Kidney (++) Gastric (++) Lung (++) Lung (++) Pancreatic (++)Pancreatic (++) Gastric (+) Liver (+) Liver (+) ST14 70% Colon TumorsColon (++) CBT026_T Cell Surface 20% Squ Lung Tumors Lung (++)CBT026_HES_Primary 80% NHL Node Pancreatic (++) 60% Ovary Tumors Gastric(+) 38% Panc Tumors XP_114346 Colon (++) Colon (++) HT29_StemCell CellSurface Lung (++) Lung (++) CBT026_N CBT026_T CBT026_HES_PrimaryFLJ14681 H1299_StemCell_Glyco Cell Surface SERPINF2 Melanoma SecretedStemcell_EGT003D DPEP1 38% Liver Colon (++) Colon (+++) CBT026_N CellSurface 20% Colon 20% Prostate 10% Gastric GP25L2 CBT026_N Cell SurfaceGPC1 Gastric (++) RKO Sphere vs Cell Surface Liver (++) Adherent PNGFBreast (+) Lung (+) gi|23268459 Melanoma Secreted StemCell_EGT003D NCSTNCBT026_N Cell Surface CBT026_T CBT026_HES_Primary CD97 CBT026_N CellSurface LY75 Colon (++) Kidney (++) HT29_Stemcell Cell Surface Kidney(++) SLC11A2 HT29_StemCell Cell Surface ANPEP H1299_StemCell_GlycoSecreted GGT1 CBT026_N Cell Surface CEACAM1 HT29_StemCell Cell SurfaceCBT026_N CBT026_T CBT026_HES_Primary PGLYRP2 Melanoma SecretedStemcell_EGT003D PZP LS123_StemCell Secreted Q9Y3B3 MelanomaStemcell_EGT003D Cell Surface BGN CBT026_2Maps Secreted CBT026_NCBT026_T CBT026_HES_Primary HT29_StemCell RKO Sphere vs Adherent PNGFH1299_Stemcell_Glyco Melanoma Stemcell_EGT003D PIGR Colon (++)HT29_Stemcell Cell Surface Lung (++) DSG2 Kidney (++) Kidney (++)HT29_Stemcell Cell Surface Pancreatic (+) gi|22859168 Colon (++)HT29_Stemcell Cell Surface Pancreatic (++) Gastric (+) Kidney (+)Melanoma (+) PTGFRN CBT026_N Cell Surface CBT026_HES_Primary IGSF8 66%NHL Node Colon (++) CBT026_HES_Primary Cell Surface 20% Ovary TumorsGastric (++) 30% Melanoma Lung (++) gi|41352831 Melanoma Cell SurfaceStemCell_EGT003D CDCP1 Breast (+++) CBT026_N Cell Surface Colon (+++)Lung (+++) Gastric (++) Liver (++) Melanoma (++) Pancreatic (++) ATP1B170% Melanoma node Lung (++) Breast (++) CBT026_N Cell Surface 70% BreastTumors Melanoma (++) Colon (++) CBT026_T 67% Glioblastoma Melanoma (++)CBT026_HES_Primary 50% Melanoma HT29_Stemcell 20% Esoph Tumors CLIC1Melanoma (++) Gastric (++) H1299_Stemcell_Cys_Only Cell Surface Melanoma(++) CBT026_N CLU 30% Breast Tumors Colon (++) CBT026_T Secreted 60%Ovary Tumors Melanoma (+) CBT026_HES_Primary 30% Prostate TumorsH1299_Stemcell_Glyco TIMP1 RKO Sphere vs Secreted Adherent PNGF TM9SF150% Panc Tumors Kidney (++) HT29_Stemcell Cell Surface 20% ProstateTumors Lung (++) LGALS3BP Melanoma Secreted Stemcell_EGT003D CBT026_TCBT026_N CBT026_HES_Primary H1299_Stem cell_Glyco FAT Gastric (++) Colon(++) CBT026_N Cell Surface Gastric (++) Liver (+) HLA-C CBT026_N CellSurface ENPP1 Colon (+) H1299_Stemcell_Cys_Only SecretedH1299_Stemcell_Glyco HSPG2 HT29_StemCell Secreted H1299_StemCell_GlycoCBT026_N PLTP Lung (+) Colon (++) CBT026_12Maps Secreted Gastric (+)Melanoma (+) PON1 RKO Sphere vs Secreted Adherent PNGF HLA-C CBT026_NCell Surface MUCDHL Colon (++) CBT026_N Cell Surface CBT026_T SLC7A1Colon (++) CSC Overlap Cell Surface Liver (++) CBT026_HES_PrimaryPancreatic (++) Gastric (+) GPR56 CBT026_N Cell Surface P2RX4 Kidney(++) Colon (++) CBT026_N Cell Surface Kidney (++) Gastric (+) Lung (+)gi|21361918 Melanoma (++) Melanoma Secreted Colon (+) StemCell_EGT003DGastric (+) HLA-A CBT026_12Maps Cell Surface ATP6V0A1 CBT026_T CellSurface CBT026_HES_Primary Cell Surface TM9SF4 40% Colon Tumors Colon(++) Melanoma Cell Surface 100% Liver Tumors Stemcell_EGT003D 56% NSCLung Tumors 64% Squ Lung Tumors 80% Melanoma Node 50% NHL Node 88% PancTumors 50% Met Panc Tumors 70% Melanoma TMED7 Melanoma Cell SurfaceStemcell_EGT003D KIAA1815 33% Glioblastoma Breast (++) CBT026_N CellSurface 38% Liver Tumors Lung (++) HT29_Stemcell 73% Squ Lung Tumors 50%Panc Tumors 30% Prostate Tumors CD82 Lung (+) Colon (++)CBT026_HES_Primary Cell Surface Ovary (+) Pancreatic (++) LAMA4CBT026_12Maps Secreted SERPINE2 Kidney (++) Lung (++)H1299_Stemcell_Glyco Lung (++) Ovary (+) GOLPH2 HT29_StemCell SecretedCBT026_N BMP1 H1299_Stemcell_Glyco Secreted EVA1 HT29_Stem cell CellSurface SERPINF1 CBT026_12 maps Secreted HT29_Stemcell CBT026_HES_Primary GREM2 HT29_Stemcell Secreted FAM38A HT29_Stemcell Cell SurfaceLIPG HT29_stem cell Secreted ST3GAL1 Melanoma (++) CBT026_N SecretedLNPEP CBT026_N Cell Surface MFAP4 CBT026_HES_Primary Secreted CBT026_NCBT026_T DKFZP564G2022 CBT026_N Cell Surface CEACAM6 Gastric (+)Prostate (+) CBT026_N Cell Surface CBT026_T CBT026_HES_primary FLJ11273Colon (+) Pancreatic (++) CBT026_T Cell Surface Colon (+) CBT026_NCBT026_HES_Primary CEACAM7 CBT026_N Cell Surface CANT1 CBT026_N SecretedSSR1 Ovary (+) Ovary (+) CBT026_N Cell Surface Pancreatic (+) CBT026_TCBT026_HES_Primary TTYH3 Colon (++) Colon (++) CBT026_N Cell SurfaceLung (++) Gastric (++) CBT026_T Melanoma (++) Liver (++)CBT026_HES_Primary Ovary (+) Lung (++) Prostate (+) Melanoma (++) Kidney(+) Pancreatic (+) CLPTM1 CBT026_N Cell Surface CBT026_HES_Primary FAM3DColon (++) CBT026_N Secreted LOC284361 Colon (++) Colon (++) CBT026_NCell Surface Lung (++) Lung (++) Kidney (+) Pancreatic (+) Liver (+)KIAA0090 Colon (++) Colon (++) CBT026_N Secreted Breast (+) Lung (++)CBT026_T Pancreatic (+) CBT026_HES_Primary LAMA5 H1299_StemCell_GlycoSecreted ATP6AP1 CBT026_HES_Primary Cell Surface PTPRK Lung (+) Colon(++) CBT026_HES_Primary Cell Surface Ovary (+) Pancreatic (++) Prostate(+) NCLN H1299_Stem Cell Surface Cell_Glyco ANGPTL4 Colon (++) Colon(++) H1299_StemCell_Glyco Secreted Kidney (++) Kidney (++) Pancreatic(++) MFGE8 H1299_StemCell_Glyco Cell Surface COMT 100% Kidney TumorsBreast (++) Colon (+++) LS123_Stemcell Cell Surface 100% Ovary TumorsColon (++) Lung (+++) 60% Melanoma Node Gastric (++) Melanoma (+++) 50%Melanoma Breast (++) 43% Panc Tumors Gastric (++) 50% Colon TumorsKidney (++) 30% Bladder Tumors Liver (++) 50% Glioblastoma Prostate (++)30% Squ Lung Tumors 30% Prostate Tumors 25% Liver Tumors GLG1 30% SquLung Tumors Liver (++) H1299_Stemcell_Glyco Cell Surface 50% MelanomaNode Breast (+) Melanoma 43% Panc Tumors Stemcell_EGT003D 80% MelanomaCD44 CBT026_HES_Primary Cell Surface LAMP1 Prostate (++) CBT026_N CellSurface CBT026_T CBT026_HES_Primary SLC3A2 100% NSC Lung TumorsPancreatic (++) Lung (+++) CBT026_HES_Primary Cell Surface 100% Squ LungTumors Colon (++) 90% Melanoma Gastric (++) 83% Glioblastoma Pancreatic(++) 80% Colon Tumors Prostate (++) 50% Breast Tumors SCARB2HT29_StemCell Cell Surface CBT026_N CBT026_T CBT026_HES_Primary ITGA3Pancreatic (++) Pancreatic (++) CBT026_N Cell Surface CBT026_HES_PrimaryPLXNB2 CBT026_N Cell Surface CBT026_HES_Primary LAMP2 Lung (++) Liver(++) CBT026_N Cell Surface Melanoma (++) Lung (++) CBT026_T Pancreatic(++) Colon (+) CBT026_HES_Primary Colon (+) AADACL1 60% Colon TumorsCBT026_N Cell Surface 80% Melanoma Node CBT026_T 33% NHL NodeCBT026_HES_Primary 100% Panc Tumors 75% Met Panc Tumors 80% MelanomaALCAM 40% Breast Tumors Colon (++) Breast (++) CBT026_five sets withCell Surface 30% Bladder Tumors Gastric (++) Colon (++) N primary 20%Colon Tumors Kidney (++) Gastric (++) Liver (++) Kidney (++) Melanoma(+) Liver (++) Pancreatic (++) Melanoma (+) SLC2A1 Colon (-) Breast (-)CBT026_T Cell Surface Pancreatic (-) Colon (-) CBT026_N Lung (-) Gastric(-) CBT026_HES_Primary Breast (-) Kidney (-) Kidney (-) Liver (-)Gastric (-) Lung (+) Prostate (-) Melanoma (-) Melanoma (-) Pancreatic(-) Liver (-) Prostate (-) ITGB1 CBT026_HES_Primary Cell Surface BSGLung (-) Lung (-) CBT026_HES_Primary Cell Surface

1. A method of inhibiting proliferation of pancreatic cancer cells invitro, the method comprising contacting the pancreatic cancer cells invitro with an isolated antibody that selectively binds to a SLC5A1protein to thereby inhibit proliferation of the pancreatic cancer cellsin vitro.
 2. The method of claim 1, wherein the amino acid sequence ofthe SLC5A1 protein comprises SEQ ID NO:191.
 3. The method of claim 1,wherein the antibody is coupled to at least one agent selected from thegroup consisting of drugs, toxins, differentiation inducers, andradionuclides.
 4. The method of claim 2, wherein the antibody is coupledto at least one agent selected from the group consisting of drugs,toxins, differentiation inducers, and radionuclides.